U.S. patent application number 11/803843 was filed with the patent office on 2008-04-03 for polymers for functional particles.
This patent application is currently assigned to Massachusetts Institute of Technology. Invention is credited to Omid C. Farokhzad, Frank X. Gu, Robert S. Langer, Benjamin A. Teply.
Application Number | 20080081074 11/803843 |
Document ID | / |
Family ID | 38603373 |
Filed Date | 2008-04-03 |
United States Patent
Application |
20080081074 |
Kind Code |
A1 |
Gu; Frank X. ; et
al. |
April 3, 2008 |
Polymers for functional particles
Abstract
The present invention generally relates to polymers and
macromolecules, in particular, to block polymers useful in
particles such as nanoparticles. One aspect of the invention is
directed to a method of developing nanoparticles with desired
properties. In one set of embodiments, the method includes
producing libraries of nanoparticles having highly controlled
properties, which can be formed by mixing together two or more
macromolecules in different ratios. One or more of the
macromolecules may be a polymeric conjugate of a moiety to a
biocompatible polymer. In some cases, the nanoparticle may contain
a drug. The moiety, in some embodiments, may have a molecular
weight greater than about 1000 Da; for example, the moiety may
include a polypeptide or a polynucleotide, such as an aptamer. The
moiety may also be a targeting moiety, an imaging moiety, a
chelating moiety, a charged moiety, or a therapeutic moiety.
Another aspect of the invention is directed to systems and methods
of producing such polymeric conjugates. In some embodiments, a
solution containing a polymer is contacted with a liquid, such as
an immiscible liquid, to form nanoparticles containing the
polymeric conjugate. Other aspects of the invention are directed to
methods using such libraries, methods of using or administering
such polymeric conjugates, methods of promoting the use of such
polymeric conjugates, kits involving such polymeric conjugates, or
the like.
Inventors: |
Gu; Frank X.; (Cambridge,
MA) ; Teply; Benjamin A.; (Omaha, NE) ;
Langer; Robert S.; (Newton, MA) ; Farokhzad; Omid
C.; (Chestnut Hill, MA) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, P.C.
600 ATLANTIC AVENUE
BOSTON
MA
02210-2206
US
|
Assignee: |
Massachusetts Institute of
Technology
Cambridge
MA
The Brigham & Women's Hospital, Inc.
Boston
MA
|
Family ID: |
38603373 |
Appl. No.: |
11/803843 |
Filed: |
May 15, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60747240 |
May 15, 2006 |
|
|
|
Current U.S.
Class: |
424/489 ;
424/94.1; 506/23; 514/772.4 |
Current CPC
Class: |
Y10T 428/2982 20150115;
A61K 47/6937 20170801; C08G 63/664 20130101; C08G 63/912 20130101;
A61P 35/00 20180101; A61K 51/1244 20130101; A61K 47/6935 20170801;
Y10S 977/773 20130101; A61K 49/00 20130101; Y10S 977/915 20130101;
Y10S 977/906 20130101; A61K 9/5153 20130101; A61K 47/549 20170801;
B82Y 5/00 20130101 |
Class at
Publication: |
424/489 ;
424/094.1; 506/023; 514/772.4 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 38/43 20060101 A61K038/43; C40B 50/00 20060101
C40B050/00; A61K 47/32 20060101 A61K047/32 |
Goverment Interests
GOVERNMENT FUNDING
[0002] Research leading to various aspects of the present invention
was sponsored, at least in part, by the National Cancer Institute,
Grant No. CA 119349, and the National Institute of Biomedical
Imaging and Bioengineering, Grant No. EB 003647. The U.S.
Government may have certain rights in the invention.
Claims
1. A composition, comprising: a particle having an average
characteristic dimension of less than about 1 micrometer, the
particle comprising a macromolecule comprising a first portion
comprising a biocompatible polymer and a second portion comprising
a moiety selected from the group consisting of a targeting moiety,
an imaging moiety, a chelating moiety, a moiety having multiple
charge groups, and a therapeutic moiety, the moiety having an
essentially nonzero concentration internally of the particle.
2. The composition of claim 1, wherein the moiety has a molecular
weight of at least about 1000 Da.
3. The composition of claim 1, wherein the moiety has a molecular
weight of no more than about 1000 Da.
4. The composition of claim 1, wherein the macromolecule is a block
copolymer.
5. The composition of claim 1, wherein the macromolecule is
amphiphilic.
6. The composition of claim 1, wherein at least a portion of the
macromolecule is biodegradable.
7. The composition of claim 1, wherein at least a portion of the
macromolecule is hydrolyzable.
8. The composition of claim 1, wherein the biocompatible polymer
comprises poly(lactide-co-glycolide).
9. The composition of claim 1, wherein the targeting moiety
comprises a polypeptide.
10. The composition of claim 1, wherein the targeting moiety
comprises an aptamer.
11. The composition of claim 1, wherein the targeting moiety
specifically binds prostate specific membrane antigen.
12. The composition of claim 1, wherein the targeting moiety
comprises an antibody or an antibody fragment.
13. The composition of claim 1, wherein the targeting moiety is
able to specifically bind to a cell surface receptor.
14. The composition of claim 1, wherein the targeting moiety is
able to specifically bind to a biological entity.
15. The composition of claim 1, wherein the particle further
comprises a second macromolecule comprising the biocompatible
polymer and is free of the moiety.
16. The composition of claim 1, wherein the particle further
comprises a drug.
17. The composition of claim 16, wherein the drug is
hydrophobic.
18. The composition of claim 1, wherein the particle comprises a
nucleic acid.
19. The composition of claim 1, wherein the particle comprises a
peptide or a protein.
20. The composition of claim 1, wherein the particle comprises
docetaxel.
21. The composition of claim 1, wherein the particle comprises an
enzyme.
22. The composition of claim 1, wherein the particle has an average
characteristic dimension of less than about 150 nm.
23. The composition of claim 1, wherein the block copolymer further
comprises a third block comprising a poly(alkylene glycol).
24. The composition of claim 23, wherein the poly(alkylene glycol)
comprises poly(ethylene glycol).
25. The composition of claim 1, wherein the particle is made by a
process comprising: providing a solution comprising the
macromolecule; and contacting the solution with a polymer
nonsolvent to produce the particle.
26. The composition of claim 25, wherein the solution comprising
the macromolecule is an organic solution and the polymer nonsolvent
is an aqueous solution.
27. A method of developing nanoparticles with desired properties,
comprising: providing a first macromolecule comprising a first
biocompatible polymer and a moiety selected from the group
consisting of a targeting moiety, an imaging moiety, a chelating
moiety, a moiety having multiple charge groups, and a therapeutic
moiety; providing a second macromolecule comprising a second
biocompatible polymer; producing a library of nanoparticles having
different ratios of the first and second macromolecules by forming
nanoparticles from mixtures comprising the first and second
macromolecules at different ratios; and identifying a nanoparticle
from the library of nanoparticles having one or more desired
properties.
28. The method of claim 27, wherein the first biocompatible polymer
is substantially the same as the second biocompatible polymer.
29. The method of claim 27, wherein the first polymer further
comprises a poly(ethylene glycol).
30. The method of claim 27, wherein the second polymer further
comprises a poly(ethylene glycol).
31. The method of claim 27, wherein the first biocompatible polymer
comprises poly(lactide-co-glycolide).
32. The method of claim 27, further comprising adding a drug prior
to forming the nanoparticles.
33. The method of claim 32, wherein the drug is doclitaxel.
34. The method of claim 27, further comprising adding an
interfering RNA prior to forming the nanoparticles.
35. The method of claim 27, further comprising adding a peptide or
a protein prior to forming the nanoparticles.
36. The method of claim 27, further comprising adding an enzyme
prior to forming the nanoparticles.
37. A method of producing a library, comprising: providing a first
macromolecule comprising a first block having a repeat unit and a
second block comprising a moiety selected from the group consisting
of a targeting moiety, an imaging moiety, a chelating moiety, a
moiety having multiple charge groups, and a therapeutic moiety;
providing a second polymer comprising the first repeat unit but not
comprising the targeting moiety; and producing a library of
nanoparticles having different ratios of the first macromolecule
and second polymer by forming nanoparticles from mixtures
comprising the first macromolecule and the second polymer at
different ratios.
38. A composition, comprising: a particle having an average
characteristic dimension of less than about 1 micrometer, the
particle comprising a first macromolecule and a second
macromolecule; wherein the first macromolecule is a block copolymer
comprising a first biocompatible polymer, a poly(alkylene glycol),
and a moiety selected from the group consisting of a targeting
moiety, an imaging moiety, a chelating moiety, a moiety having
multiple charge groups, and a therapeutic moiety; and wherein the
second macromolecule is a block copolymer comprising a
poly(alkylene glycol) and a second biocompatible polymer
distinguishable from the first biocompatible polymer.
39. A composition, comprising: a particle having an average
characteristic dimension of less than about 1 micrometer, the
particle having a surface comprising a first macromolecule and a
second macromolecule, the first macromolecule comprising a first
poly(alkylene glycol) chain having a first length and a moiety
selected from the group consisting of a targeting moiety, an
imaging moiety, a chelating moiety, a moiety having multiple charge
groups, and a therapeutic moiety, and the second macromolecule
comprising a second poly(alkylene glycol) chain having a second
length different from the first length.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 60/747,240, filed May 15, 2006,
entitled "Multi-Block Co-Polymers for Development of Functional
Particles," by Farokhzad, et al., incorporated herein by
reference.
FIELD OF INVENTION
[0003] The present invention generally relates to polymers and
macromolecules and, in particular, to block polymers useful in
particles such as nanoparticles.
BACKGROUND
[0004] The delivery of a drug to a patient with controlled-release
of the active ingredient has been an active area of research for
decades and has been fueled by the many recent developments in
polymer science and the need to deliver more labile pharmaceutical
agents such as nucleic acids, proteins, and peptides. In addition,
controlled release polymer systems can be designed to provide a
drug level in the optimum range over a longer period of time than
other drug delivery methods, thus increasing the efficacy of the
drug and minimizing problems with patient compliance.
[0005] Biodegradable particles have been developed as sustained
release vehicles used in the administration of small molecule drugs
as well as protein and peptide drugs and nucleic acids. The drugs
are typically encapsulated in a polymer matrix which is
biodegradable and biocompatible. As the polymer is degraded and/or
as the drug diffuses out of the polymer, the drug is released into
the body. Typically, polymers used in preparing these particles are
polyesters such as poly(lactide-co-glycolide) (PLGA), polyglycolic
acid, poly-beta-hydroxybutyrate, polyacrylic acid ester, etc. These
particles can also protect the drug from degradation by the body.
Furthermore, these particles can be administered using a wide
variety of administration routes.
[0006] Targeting controlled release polymer systems (e.g., targeted
to a particular tissue or cell type or targeted to a specific
diseased tissue but not normal tissue) is desirable because it
reduces the amount of a drug present in tissues of the body that
are not targeted. This is particularly important when treating a
condition such as cancer where it is desirable that a cytotoxic
dose of the drug is delivered to cancer cells without killing the
surrounding non-cancerous tissue. Effective drug targeting should
reduce the undesirable and sometimes life threatening side effects
common in anticancer therapy.
SUMMARY OF THE INVENTION
[0007] The present invention generally relates to polymers and
macromolecules and, in particular, to block polymers useful in
particles such as nanoparticles. The subject matter of the present
invention involves, in some cases, interrelated products,
alternative solutions to a particular problem, and/or a plurality
of different uses of one or more systems and/or articles.
[0008] In one aspect, the present invention is directed to a
method. In one set of embodiments, the method is a method of
developing nanoparticles with desired properties. According to a
first embodiment, the method includes acts of providing a first
macromolecule comprising a first biocompatible polymer and a moiety
selected from the group consisting of a targeting moiety, an
imaging moiety, a chelating moiety, a moiety having multiple charge
groups, and a therapeutic moiety; providing a second macromolecule
comprising a second biocompatible polymer; producing a library of
nanoparticles having different ratios of the first and second
macromolecules by forming nanoparticles from mixtures comprising
the first and second macromolecules at different ratios; and
identifying a nanoparticle from the library of nanoparticles having
one or more desired properties.
[0009] The method, in another embodiment, includes acts of
providing a first macromolecule comprising a first block having a
repeat unit and a second block comprising a moiety selected from
the group consisting of a targeting moiety, an imaging moiety, a
chelating moiety, a moiety having multiple charge groups, and a
therapeutic moiety; providing a second polymer comprising the first
repeat unit but not comprising the targeting moiety; and producing
a library of nanoparticles having different ratios of the first
macromolecule and second polymer by forming nanoparticles from
mixtures comprising the first macromolecule and the second polymer
at different ratios.
[0010] In yet another embodiment, the method includes acts of
providing a first biocompatible hydrophobic polymer; providing a
second biocompatible hydrophilic polymer; providing a moiety
selected from the group consisting of a targeting moiety, an
imaging moiety, a chelating moiety, a moiety having multiple charge
groups, and a therapeutic moiety; reacting the first biocompatible
hydrophobic polymer, the second biocompatible hydrophilic polymer,
and the moiety to produce a macromolecule; producing a library of
nanoparticles comprising the macromolecule and at least one other
polymer by forming nanoparticles from mixtures comprising the
macromolecule and the at least one other polymer at different
ratios; and identifying a nanoparticle from the library of
nanoparticles having one or more desired properties
[0011] In accordance with still another embodiment, the method
includes acts of providing a biocompatible hydrophilic polymer;
providing a moiety selected from the group consisting of a
targeting moiety, an imaging moiety, a chelating moiety, a moiety
having multiple charge groups, and a therapeutic moiety; reacting
the biocompatible hydrophilic polymer and the moiety to produce a
macromolecule; producing a library of nanoparticles comprising the
macromolecule and at least one other polymer by forming
nanoparticles from mixtures comprising the macromolecule and the at
least one other polymer at different ratios; and identifying a
nanoparticle from the library of nanoparticles having one or more
desired properties.
[0012] The method, in one embodiment, includes acts of providing a
biocompatible hydrophobic polymer; providing a moiety selected from
the group consisting of a targeting moiety, an imaging moiety, a
chelating moiety, a moiety having multiple charge groups, and a
therapeutic moiety; reacting the biocompatible hydrophobic polymer
and the moiety to produce a macromolecule; producing a library of
nanoparticles comprising the macromolecule and at least one other
polymer by forming nanoparticles from mixtures of the macromolecule
and the at least one other polymer at different ratios; and
identifying a nanoparticle from the library of nanoparticles having
one or more desired properties.
[0013] In another set of embodiments, the method includes acts of
providing a first macromolecule comprising a biocompatible
hydrophobic polymer, a biocompatible hydrophilic polymer, and a
moiety selected from the group consisting of a targeting moiety, an
imaging moiety, a chelating moiety, a moiety having multiple charge
groups, and a therapeutic moiety; providing a second macromolecule
comprising the biocompatible hydrophobic polymer, the second
macromolecule having a polymeric portion distinguishable from the
polymer portion of the first macromolecule; producing a library of
nanoparticles having different ratios of the first and second
macromolecules by forming nanoparticles from mixtures of the first
and second macromolecules at different ratios; and identifying a
nanoparticle from the library of nanoparticles having one or more
desired properties.
[0014] In still another set of embodiments, the method includes
acts of providing a first macromolecule comprising a biocompatible
hydrophobic polymer, a biocompatible hydrophilic polymer, and a
moiety selected from the group consisting of a targeting moiety, an
imaging moiety, a chelating moiety, a moiety having multiple charge
groups, and a therapeutic moiety; providing a second macromolecule
comprising the biocompatible hydrophobic polymer and the
biocompatible hydrophilic polymer, the second macromolecule having
a polymeric portion distinguishable from the polymer portion of the
first macromolecule; producing a library of nanoparticles having
different ratios of the first and second macromolecules by forming
nanoparticles from mixtures of the first and second macromolecules
at different ratios; and identifying a nanoparticle from the
library of nanoparticles having one or more desired properties.
[0015] According to yet another embodiment, the method includes
acts of providing a first macromolecule comprising a biocompatible
hydrophobic polymer and a biocompatible hydrophilic polymer;
providing a second macromolecule comprising the biocompatible
hydrophobic polymer and a moiety selected from the group consisting
of a targeting moiety, an imaging moiety, a chelating moiety, a
moiety having multiple charge groups, and a therapeutic moiety, the
second macromolecule having a polymeric portion distinguishable
from the polymer portion of the first macromolecule; producing a
library of nanoparticles having different ratios of the first and
second macromolecules by forming nanoparticles from mixtures of the
first and second macromolecules at different ratios; and
identifying a nanoparticle from the library of nanoparticles having
one or more desired properties.
[0016] In another set of embodiments, the method is a method of
producing a library. According to one embodiment, the method
includes acts of providing a first block copolymer comprising a
biocompatible polymer, a poly(alkylene glycol), and a moiety
selected from the group consisting of a targeting moiety, an
imaging moiety, a chelating moiety, a moiety having multiple charge
groups, and a therapeutic moiety; providing a second polymer
comprising a block copolymer comprising a biocompatible polymer and
a poly(alkylene glycol) and having a polymeric portion
distinguishable from the polymeric portion of the first polymer;
and producing a library of nanoparticles having different ratios of
the first and second polymers by forming nanoparticles from
mixtures of the first and second polymers at different ratios.
[0017] According to another embodiment, the method includes acts of
providing a first block copolymer comprising first and second
polymers; providing a second block copolymer comprising the first
and second polymers and further comprising a moiety selected from
the group consisting of a targeting moiety, an imaging moiety, a
chelating moiety, a moiety having multiple charge groups, and a
therapeutic moiety; and producing a library of nanoparticles having
different ratios of the first and second polymers by forming
nanoparticles from mixtures of the first and second polymers at
different ratios.
[0018] The method, in one set of embodiments, includes acts
providing an solution comprising an amphiphilic macromolecule
comprising a repeat unit and a moiety selected from the group
consisting of a targeting moiety, an imaging moiety, a chelating
moiety, a moiety having multiple charge groups, and a therapeutic
moiety; and contacting the solution with a polymer nonsolvent to
produce particles comprising the amphiphilic macromolecule, the
particles having an average characteristic dimension of less than
about 1 micrometer. In another set of embodiments, the method
includes acts of providing a solution comprising a first block
copolymer and a second block copolymer; and contacting the solution
with a polymer nonsolvent to produce particles particle having an
average characteristic dimension of less than about 1 micrometer.
In still another set of embodiments, the method includes acts of
providing a solution comprising a polymer comprising a
biocompatible polymer, a poly(alkylene glycol), and a moiety
selected from the group consisting of a targeting moiety, an
imaging moiety, a chelating moiety, a moiety having multiple charge
groups, and a therapeutic moiety; and contacting the solution with
a polymer nonsolvent to produce particles having an average
characteristic dimension of less than about 1 micrometer.
[0019] In accordance with yet another set of embodiments, the
method includes an act of reacting a carboxylic acid-terminated
poly(ester-ether) copolymer with a moiety selected from the group
consisting of a targeting moiety, an imaging moiety, a chelating
moiety, a moiety having multiple charge groups, and a therapeutic
moiety, the moiety comprising an amine, without using
N-hydroxysuccinimide to produce a block copolymer.
[0020] The method, in still another set of embodiments, includes in
a single reaction, an act of reacting a carboxylic acid-terminated
poly(ester-ether) copolymer with a moiety selected from the group
consisting of a targeting moiety, an imaging moiety, a chelating
moiety, a moiety having multiple charge groups, and a therapeutic
moiety, the targeting moiety comprising an amine, to produce a
block copolymer.
[0021] In another aspect, the present invention is directed to a
composition. In one set of embodiments, the composition comprises a
particle having an average characteristic dimension of less than
about 1 micrometer, where the particle comprises a macromolecule
comprising a first portion comprising a biocompatible polymer and a
second portion comprising a moiety selected from the group
consisting of a targeting moiety, an imaging moiety, a chelating
moiety, a moiety having multiple charge groups, and a therapeutic
moiety. In some cases, the moiety has an essentially nonzero
concentration internally of the particle.
[0022] According to another set of embodiments, the composition
includes a particle having an average characteristic dimension of
less than about 1 micrometer, where the particle includes a first
macromolecule and a second macromolecule. In some cases, the first
macromolecule is a block copolymer comprising a first biocompatible
polymer, a poly(alkylene glycol), and a moiety selected from the
group consisting of a targeting moiety, an imaging moiety, a
chelating moiety, a moiety having multiple charge groups, and a
therapeutic moiety. In certain instances, the second macromolecule
is a block copolymer comprising a poly(alkylene glycol) and a
second biocompatible polymer distinguishable from the first
biocompatible polymer
[0023] In yet another set of embodiments, the composition includes
a particle having an average characteristic dimension of less than
about 1 micrometer, where the particle has a surface comprising a
first macromolecule and a second macromolecule. In some cases, the
first macromolecule comprises a first poly(alkylene glycol) chain
having a first length and a moiety selected from the group
consisting of a targeting moiety, an imaging moiety, a chelating
moiety, a moiety having multiple charge groups, and a therapeutic
moiety, and the second macromolecule comprises a second
poly(alkylene glycol) chain having a second length different from
the first length.
[0024] The composition, in accordance with one set of embodiments,
includes a particle, having an average characteristic dimension of
less than about 1 micrometer, comprising a first macromolecule
comprising a first biocompatible polymer and a moiety selected from
the group consisting of a targeting moiety, an imaging moiety, a
chelating moiety, a moiety having multiple charge groups, and a
therapeutic moiety, and a second macromolecule comprising a second
biocompatible polymer. In some cases, the particle is chosen from a
library of nanoparticles having different ratios of the first and
second macromolecules.
[0025] In another set of embodiments, the composition includes a
particle, having an average characteristic dimension of less than
about 1 micrometer, comprising a macromolecule comprising a first
biocompatible hydrophobic polymer, a second biocompatible
hydrophilic polymer, and a moiety selected from the group
consisting of a targeting moiety, an imaging moiety, a chelating
moiety, a moiety having multiple charge groups, and a therapeutic
moiety. The particle may further comprise at least one other
polymer. The particle, in some cases, may be chosen from a library
of nanoparticles having different ratios of the macromolecule and
the at least one other polymer.
[0026] In yet another set of embodiments, the composition includes
a particle, having an average characteristic dimension of less than
about 1 micrometer, comprising a macromolecule comprising a
biocompatible hydrophilic polymer and a moiety selected from the
group consisting of a targeting moiety, an imaging moiety, a
chelating moiety, a moiety having multiple charge groups, and a
therapeutic moiety. In some cases, the particle further comprises
at least one other polymer. In addition, the particle may be chosen
from a library of nanoparticles having different ratios of the
macromolecule and the at least one other polymer.
[0027] The composition, in still another set of embodiments,
includes a particle, having an average characteristic dimension of
less than about 1 micrometer, comprising a macromolecule comprising
a biocompatible hydrophobic polymer and a moiety selected from the
group consisting of a targeting moiety, an imaging moiety, a
chelating moiety, a moiety having multiple charge groups, and a
therapeutic moiety. In one embodiment, the particle further
comprising at least one other polymer, and in some cases, the
particle is chosen from a library of nanoparticles having different
ratios of the macromolecule and the at least one other polymer.
[0028] In one set of embodiments, the composition includes a
particle, having an average characteristic dimension of less than
about 1 micrometer, comprising a first macromolecule comprising a
biocompatible hydrophobic polymer, a biocompatible hydrophilic
polymer, and a moiety selected from the group consisting of a
targeting moiety, an imaging moiety, a chelating moiety, a moiety
having multiple charge groups, and a therapeutic moiety, and a
second macromolecule comprising the biocompatible hydrophobic
polymer. The second macromolecule may have, in some cases, a
polymeric portion distinguishable from the polymer portion of the
first macromolecule. In certain instances, the particle is chosen
from a library of nanoparticles having different ratios of the
first and second macromolecules.
[0029] In another set of embodiments, the composition includes a
particle, having an average characteristic dimension of less than
about 1 micrometer, comprising a first macromolecule comprising a
biocompatible hydrophobic polymer, a biocompatible hydrophilic
polymer, and a moiety selected from the group consisting of a
targeting moiety, an imaging moiety, a chelating moiety, a moiety
having multiple charge groups, and a therapeutic moiety, and a
second macromolecule comprising the biocompatible hydrophobic
polymer and the biocompatible hydrophilic polymer. In one
embodiment, the second macromolecule has a polymeric portion
distinguishable from the polymer portion of the first
macromolecule. In some cases, the particle is chosen from a library
of nanoparticles having different ratios of the first and second
macromolecules.
[0030] The composition, in yet another set of embodiments, includes
a particle, having an average characteristic dimension of less than
about 1 micrometer, comprising a first macromolecule comprising a
biocompatible hydrophobic polymer and a biocompatible hydrophilic
polymer, and a second macromolecule comprising the biocompatible
hydrophobic polymer and a moiety selected from the group consisting
of a targeting moiety, an imaging moiety, a chelating moiety, a
moiety having multiple charge groups, and a therapeutic moiety. In
some cases, the second macromolecule has a polymeric portion
distinguishable from the polymer portion of the first
macromolecule. In one embodiment, the particle is chosen from a
library of nanoparticles having different ratios of the first and
second macromolecules.
[0031] In still another set of embodiments, the composition
includes a particle having an average characteristic dimension of
less than about 1 micrometer, comprising a polymer comprising a
first component comprising a biocompatible polymer suitable for
drug encapsulation, a second component comprising a polymeric
material for decreasing immunogenicity, and a third component
comprising a moiety selected from the group consisting of a
targeting moiety, an imaging moiety, a chelating moiety, a moiety
having multiple charge groups, and a therapeutic moiety. In one
embodiment, the third component has an essentially nonzero
concentration internally of the particle.
[0032] According to yet another set of embodiments, the composition
includes a particle having an average characteristic dimension of
less than about 1 micrometer, produced using a macromolecule
comprising a first component comprising a biocompatible polymer
suitable for drug encapsulation, a second component comprising a
polymeric material for decreasing immunogenicity, and a third
component comprising a moiety selected from the group consisting of
a targeting moiety, an imaging moiety, a chelating moiety, a moiety
having multiple charge groups, and a therapeutic moiety. In one
embodiment, the particle is produced by a method comprising
providing a solution comprising the macromolecule, and contacting
the solution with a polymer nonsolvent to produce the particle.
[0033] In another set of embodiments, the composition includes a
particle having an average characteristic dimension of less than
about 1 micrometer, comprising a biocompatible polymer and a moiety
selected from the group consisting of a targeting moiety, an
imaging moiety, a chelating moiety, a moiety having multiple charge
groups, and a therapeutic moiety. In some cases, the moiety is
contained within the particle and covalently bonded to the
biocompatible polymer.
[0034] In another aspect, the present invention is directed to a
method of making one or more of the embodiments described herein,
for example, nanoparticles such as those described herein. In
another aspect, the present invention is directed to a method of
using one or more of the embodiments described herein, for example,
nanoparticles such as those described herein.
[0035] Other advantages and novel features of the present invention
will become apparent from the following detailed description of
various non-limiting embodiments of the invention when considered
in conjunction with the accompanying figures. In cases where the
present specification and a document incorporated by reference
include conflicting and/or inconsistent disclosure, the present
specification shall control. If two or more documents incorporated
by reference include conflicting and/or inconsistent disclosure
with respect to each other, then the document having the later
effective date shall control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Non-limiting embodiments of the present invention will be
described by way of example with reference to the accompanying
figures, which are schematic and are not intended to be drawn to
scale. In the figures, each identical or nearly identical component
illustrated is typically represented by a single numeral. For
purposes of clarity, not every component is labeled in every
figure, nor is every component of each embodiment of the invention
shown where illustration is not necessary to allow those of
ordinary skill in the art to understand the invention. In the
figures:
[0037] FIG. 1 is a schematic diagram illustrating a method of
producing libraries of nanoparticles having highly controlled
properties, in accordance with one embodiment of the invention;
[0038] FIG. 2A-2C illustrate schematic diagrams of various polymers
of certain embodiments of the present invention, useful for
producing particles;
[0039] FIGS. 3A-3D illustrate schematic diagrams of various
polymeric particles of certain embodiments of the present
invention;
[0040] FIGS. 4A-4C illustrate schematic diagrams of various
polymeric particles comprising two or more polymers, according to
another embodiment of the invention;
[0041] FIG. 5 is a schematic diagram a nanoparticle formed in
accordance with another embodiment of the invention;
[0042] FIG. 6 is a schematic diagram illustrating a nanoparticle
having aptamers, in yet another embodiment of the invention;
[0043] FIGS. 7A-7B illustrate in vitro activity of an
aptamer-containing nanoparticle, produced in still another
embodiment of the invention;
[0044] FIGS. 8A-8C illustrate the in vitro targeting of an
aptamer-containing nanoparticle, produced in yet another embodiment
of the invention;
[0045] FIGS. 9A-9B illustrate the determination of the amount of
aptamer on the nanoparticle surfaces, in still another embodiment
of the invention;
[0046] FIG. 10 illustrates the relationship between nanoparticle
size and molecular weight, in another embodiment of the invention;
and
[0047] FIGS. 11A-11D in vitro targeting of aptamer-containing
nanoparticles, according to still another embodiment of the
invention.
DETAILED DESCRIPTION
[0048] The present invention generally relates to polymers and
macromolecules, in particular, to block polymers useful in
particles such as nanoparticles. One aspect of the invention is
directed to a method of developing nanoparticles with desired
properties. In one set of embodiments, the method includes
producing libraries of nanoparticles having highly controlled
properties, which can be formed by mixing together two or more
macromolecules in different ratios. One or more of the
macromolecules may be a polymeric conjugate of a moiety to a
biocompatible polymer. In some cases, the nanoparticle may contain
a drug. The moiety, in some embodiments, may have a molecular
weight greater than about 1000 Da; for example, the moiety may
include a polypeptide or a polynucleotide, such as an aptamer. The
moiety may also be a targeting moiety, an imaging moiety, a
chelating moiety, a moiety having multiple charge groups, or a
therapeutic moiety. Another aspect of the invention is directed to
systems and methods of producing such polymeric conjugates. In some
embodiments, a solution containing a polymer is contacted with a
liquid, such as an immiscible liquid, to form nanoparticles
containing the polymeric conjugate. Other aspects of the invention
are directed to methods using such libraries, methods of using or
administering such polymeric conjugates, methods of promoting the
use of such polymeric conjugates, kits involving such polymeric
conjugates, or the like.
[0049] As mentioned, one aspect of the invention is directed to a
method of developing nanoparticles with desired properties, such as
desired chemical, biological, or physical properties. In one set of
embodiments, the method includes producing libraries of
nanoparticles having highly controlled properties, which can be
formed by mixing together two or more macromolecules in different
ratios. By mixing together two or more different macromolecules in
different ratios and producing particles from the macromolecules,
particles having highly controlled properties may be formed. For
example, one macromolecule may include a moiety such as a targeting
moiety, an imaging moiety, a chelating moiety, a moiety having
multiple charge groups, a neutral moiety, or a therapeutic moiety
(as discussed in detail below), while another macromolecule may be
chosen for its biocompatibility and/or its ability to control
immunogenicity of the resultant particle. In some cases, one or
more of these macromolecules may be copolymers, as discussed
below.
[0050] By creating a library of such particles, particles having
any desirable properties may be identified. For example, properties
such as surface functionality, surface charge, size, zeta (.zeta.)
potential, hydrophobicity, ability to control immunogenicity, and
the like, may be highly controlled. For instance, a library of
particles may be synthesized, and screened to identify the
particles having a particular ratio of polymers or macromolecules
that allows the particles to have a specific density of moieties
(e.g., therapeutic moieties) present on the surface of the
particle. This allows particles having one or more specific
properties to be prepared, for example, a specific size and a
specific surface density of moieties, without an undue degree of
effort. Accordingly, certain embodiments of the invention are
directed to screening techniques using such libraries, as well as
any particles identified using such libraries. In addition,
identification may occur by any suitable method. For instance, the
identification may be direct or indirect, or proceed quantitatively
or qualitatively.
[0051] As a specific, non-limiting example, one embodiment is shown
schematically in FIG. 1. In this figure, a first polymer (PLGA-PEG,
poly(lactide-co-glycolide) and poly(ethylene glycol)) is conjugated
to a targeting moiety (an aptamer, "Apt") to form a PLGA-PEG-Apt
macromolecule. The first macromolecule is mixed with a second
macromolecule (PLGA-PEG in this example) at varying ratios to form
a series of particles having different properties, for example,
different surface densities of aptamer as shown in this example.
For example, by controlling parameters such as PLGA molecular
weight, the molecular weight of PEG, the aptamer surface density,
and the nanoparticle surface charge, very precisely controlled
particles may be obtained.
[0052] More generally, the polymers or macromolecules chosen to be
used to create the library of particles may be any of a wide
variety of polymers or macromolecules, such as described in detail
below. Generally, two, three, four, or more polymers and/or
macromolecules are mixed, in a wide range of ratios (e.g., each
ranging from 0% to 100%), to form particles such as nanoparticles
having different ratios of each of the polymers or macromolecules.
The two or more macromolecules may be distinguishable in some
fashion, e.g., having different polymeric groups, having the same
polymeric groups but with different molecular weights, having some
polymeric groups in common but having others that are different
(e.g., one may have a polymeric group that the other does not
have), having the same polymeric groups but in different orders,
etc. The library of particles may have any number of members, for
example, the library may have 2, 3, 5, 10, 30, 100, 300, 1000,
3000, 10,000, 30,000, 100,000, etc. members, which can be
identified in some fashion. In some cases, the library may exist
contemporaneously; for example, the library may be contained in one
or more microtiter plates, vials, etc., or in some embodiments, the
library may have include members created at different times.
[0053] The library of particles can then be screened in some
fashion to identify those particles having one or more desired
properties, for example, surface functionality, surface charge,
size, zeta (.zeta.) potential, hydrophobicity, ability to control
immunogenicity, and the like. One or more of the macromolecules
within the particles may include one or more polymers chosen to be
biocompatible or biodegradable, one or more polymers chosen to
reduce immunogenicity, and/or one or more moieties, for instance, a
targeting moiety, an imaging moiety, a chelating moiety, a moiety
having multiple charge groups, a neutral moiety, or a therapeutic
moiety. These are discussed in detail below. The macromolecules
within the library may comprise some or all of these polymers, in
any suitable combination (including, but not limited to,
combinations in which a first polymer comprises all of these
species and a second polymer does not contain any of these
species).
[0054] As a specific example, in one embodiment, the particles may
include a first macromolecule comprising a biocompatible polymer,
and a moiety selected from the group consisting of a targeting
moiety, an imaging moiety, a chelating moiety, a moiety having
multiple charge groups, and a therapeutic moiety, and a second
macromolecule comprising a biocompatible polymer, which may or may
not be the same as that of the first macromolecule. As another
example, a first macromolecule may be a block copolymer comprising
a biocompatible hydrophobic polymer, a biocompatible hydrophilic
polymer, and a moiety selected from the group consisting of a
targeting moiety, an imaging moiety, a chelating moiety, a moiety
having multiple charge groups, a neutral moiety, and a therapeutic
moiety; and a second macromolecule distinguishable from the first
macromolecule in some fashion. For instance, the second
macromolecule may comprise the same (or a different) biocompatible
hydrophobic polymer and the same (or a different) biocompatible
hydrophilic polymer, but a different moiety (or no moiety at all)
than the first macromolecule. As another example, the first
macromolecule may comprise a biocompatible hydrophilic polymer and
a moiety selected from the group consisting of a targeting moiety,
an imaging moiety, a chelating moiety, a moiety having multiple
charge groups, a neutral moiety, and a therapeutic moiety, and a
second macromolecule distinguishable from the first macromolecule
in some fashion; or the first macromolecule may comprise a
biocompatible hydrophobic polymer and a moiety selected from the
group consisting of a targeting moiety, an imaging moiety, a
chelating moiety, a moiety having multiple charge groups, a neutral
moiety, and a therapeutic moiety, and a second macromolecule
distinguishable from the first macromolecule in some fashion.
[0055] The first macromolecule may also contain, as another
example, a first polymer comprising a biocompatible hydrophobic
polymer, a biocompatible hydrophilic polymer, and a moiety selected
from the group consisting of a targeting moiety, an imaging moiety,
a chelating moiety, a moiety having multiple charge groups, a
neutral moiety, and a therapeutic moiety, and a second
macromolecule that is distinguishable from the first macromolecule.
For instance, the second macromolecule may contain none of the
polymers of the first macromolecule, the second macromolecule may
contain one or more polymers of the first macromolecule and one or
more polymers not present in the first macromolecule, the second
macromolecule may lack one or more of the polymers of the first
macromolecule, the second macromolecule may contain all of the
polymers of the first macromolecule, but in a different order
and/or with one or more of the polymers having different molecular
weights, etc.
[0056] As yet another example, the first macromolecule may
comprising a biocompatible hydrophobic polymer, a biocompatible
hydrophilic polymer, and a moiety selected from the group
consisting of a targeting moiety, an imaging moiety, a chelating
moiety, a moiety having multiple charge groups, a neutral moiety,
and a therapeutic moiety, and the second macromolecule may comprise
the biocompatible hydrophobic polymer and the biocompatible
hydrophilic polymer, and be distinguishable from the first
macromolecule in some fashion. As still another example, the first
macromolecule may comprise a biocompatible hydrophobic polymer and
a biocompatible hydrophilic polymer, and the second macromolecule
may comprise the biocompatible hydrophobic polymer and a moiety
selected from the group consisting of a targeting moiety, an
imaging moiety, a chelating moiety, a moiety having multiple charge
groups, a neutral moiety, and a therapeutic moiety, where the
second macromolecule is distinguishable from the first
macromolecule in some fashion.
[0057] Referring now to FIGS. 2-4, non-limiting examples of various
libraries of the present invention are shown. In FIGS. 2A-2C,
examples of the synthesis of various macromolecules useful in
libraries of the present invention are shown. In FIG. 2A, a block
copolymer comprising three components is illustrated: a
biocompatible hydrophobic polymer, a biocompatible hydrophilic
polymer, and a moiety, which may be a targeting moiety, an imaging
moiety, a therapeutic moiety, a moiety having multiple charge
groups, a neutral moiety, or the like. Examples of targeting
moieties include, but are not limited to, a polynucleotide, a
polypeptide, a polysaccharide, a fatty acid, a lipid, a small
molecule, or an antibody. Examples of imaging moieties include, but
are not limited to, a fluorescent molecule, a radioactive molecule
(e.g., comprising a radioisotope), a contrast agent, a lithographic
agent, an agent sensitive to ultraviolet light, or an agent
sensitive to visible light. Examples of therapeutic agents include,
but are not limited to, a chemotherapeutic agent, a radioactive
agent, a nucleic acid-based agent, a lipid-based agent, a
carbohydrate based agent, a natural small molecule, or a synthetic
small molecule. Examples of charged molecules include cationic
molecules, or anionic molecules. In some cases, the moiety may be
an uncharged (i.e., neutral) or zwitterionic. In one embodiment,
the moiety is a moiety having multiple charge groups, for example,
a zwitterionic molecule, a molecule having multiply charged
portions, etc. In another embodiment, the moiety is divalent or a
polyvalent.
[0058] It should be noted that the macromolecules need not include
each of the components described above. For instance, as is
illustrated in FIG. 2B, a polymer useful in a library of the
present invention may include a hydrophilic biocompatible polymer
and a moiety, which may be a targeting moiety, an imaging moiety, a
therapeutic moiety, a moiety having multiple charge groups, or a
neutral moiety, i.e., the polymer need not include a biocompatible
hydrophobic polymer. Similarly, as is illustrated in FIG. 2C, a
polymer useful in a library of the present invention may include a
hydrophobic biocompatible polymer and a moiety, which may be a
targeting moiety, an imaging moiety, a therapeutic moiety, a moiety
having multiple charge groups, or a neutral moiety, i.e., the
polymer need not include a biocompatible hydrophilic polymer.
[0059] The polymers or macromolecules may then be formed into a
particle, using techniques such as those discussed in detail below.
The geometry formed by the particle from the polymer or
macromolecule may depend on factors such as the polymers that form
the particle. In addition, also as discussed below, in some cases,
the particle may include a hydrophilic agent or a hydrophobic
agent, depending on the structure of the particle. For example, the
particle may contain a drug or other therapeutic agent. The
hydrophilic or hydrophobic agent may be incorporated in the
particle during formation of the particle, e.g., by including the
agent in a solution containing the polymers that are used to form
the particle, and/or the agent may be incorporated in the particle
after its formation. Examples of such particles are shown in FIG.
3A for various moieties such as a targeting moiety, an imaging
moiety, a therapeutic moiety, a moiety having multiple charge
groups, or a neutral moiety.
[0060] In addition, the particle may contain additional polymers or
macromolecules, which may be distinguishable from the polymers or
macromolecules discussed above. Non-limiting examples are shown in
FIGS. 3B-3D. In FIG. 3B, a first macromolecule comprising a
biocompatible hydrophobic polymer, a biocompatible hydrophilic
polymer, and a moiety, which may be a targeting moiety, an imaging
moiety, a therapeutic moiety, a moiety having multiple charge
groups, or a neutral moiety, is combined with a second
macromolecule comprising a biocompatible hydrophobic polymer, to
form a particle of the preset invention. The biocompatible
hydrophobic polymer of the second macromolecule may or may not be
the same as the biocompatible hydrophobic polymer of the first
macromolecule (e.g., the second biocompatible hydrophobic polymer
may have a different molecular structure, or the same molecular
structure but the same or a different molecular weight, as the
first biocompatible hydrophobic polymer). As previously discussed,
the first and second macromolecules may be combined together at
different ratios to produce particles comprising the first and
second macromolecules.
[0061] Similarly, as is depicted in FIG. 3C, a particle of the
invention may comprise a first macromolecule comprising a
biocompatible hydrophobic polymer, a biocompatible hydrophilic
polymer, and a moiety, which may be a targeting moiety, an imaging
moiety, a therapeutic moiety, a moiety having multiple charge
groups, or a neutral moiety, and a second macromolecule that
comprises a biocompatible hydrophobic polymer and a biocompatible
hydrophilic polymer, but does not contain the moiety of the first
macromolecule (i.e., the second macromolecule may comprise a
different moiety, or no moiety at all, as is shown in FIG. 3C).
Similar to the above, the first and second macromolecules may be
combined together at different ratios to produce particles
comprising the first and second macromolecules. In FIG. 3D, as
another embodiment, a first macromolecule comprising a
biocompatible hydrophobic polymer and a biocompatible hydrophilic
polymer, is combined with a second macromolecule comprising a
biocompatible hydrophobic polymer and a moiety, which may be a
targeting moiety, an imaging moiety, a therapeutic moiety, a moiety
having multiple charge groups, or a neutral moiety. The
biocompatible hydrophobic polymer of the first macromolecule may or
may not be the same as the biocompatible hydrophobic polymer of the
second macromolecule. For instance, the two hydrophobic polymers
may have different molecular structures, or the same molecular
structures but the same or different molecular weights.
[0062] FIG. 4 illustrates that libraries can be produced using
polymers such as those described above. For example, in FIG. 4A,
polymeric particles comprising a first macromolecule comprising a
biocompatible hydrophobic polymer, a biocompatible hydrophilic
polymer, and a moiety, which may be a targeting moiety, an imaging
moiety, a therapeutic moiety, a moiety having multiple charge
groups, or a neutral moiety, and a second macromolecule that
comprises a biocompatible hydrophobic polymer and a biocompatible
hydrophilic polymer (e.g., as discussed in FIG. 3C) may be used to
create a library of particles having different ratios of the first
and second macromolecules. Such a library may be useful in
achieving particles having any number of desirable properties, for
instance properties such as surface functionality, surface charge,
size, zeta (.zeta.) potential, hydrophobicity, ability to control
immunogenicity, or the like.
[0063] In FIG. 4A, different ratios of the first and second
macromolecules (including ratios where one of the macromolecules is
absent) are combined to produce particles that form the basis of
the library. For instance, as shown in FIG. 4A, as the amount of
the first macromolecule is increased, relative to the second
macromolecule, the amount of moiety (e.g., a targeting moiety)
present on the surface of the particle may be increased. Thus, any
suitable concentration of moiety on the surface may be achieved
simply by controlling the ratio of the first and second
macromolecules in the particles. Accordingly, such a library of
particles may be useful in selecting or identifying particles
having a particular functionality.
[0064] FIGS. 4B and 4C illustrates embodiments in which more than
one type of macromolecule is used. For instance, in some cases, the
particle may include more than one type of moiety, for instance,
more than one type of therapeutic moiety, a therapeutic moiety and
an imaging moiety, etc. Any of polymeric systems herein, such as
those previously described with reference to FIGS. 2 and 3, may be
used. Particles having such moieties present on the surface of the
particle, and in any suitable concentration, may be created through
the use of a library of such particles, created as described above.
In addition, the overall concentration of moieties present on the
surface may also be controlled, for instance, by the use of
polymers that do not contain any moieties (i.e., "block AB" in
FIGS. 4A and 4B). Thus, a wide range of moieties may be presented
on the surface of the particles at any suitable concentration, and
each concentration may be independently controlled. In addition,
more than two polymers or macromolecules may be used in certain
embodiments of the invention. For example, the library may have
three, four, or more polymers or macromolecules, in which the
ratios of each are independently controlled.
[0065] As specific examples, in some embodiments of the present
invention, the library includes particles comprising polymeric
conjugates of a biocompatible polymer and a moiety selected from a
targeting moiety, an imaging moiety, a chelating moiety, a moiety
having multiple charge groups, a neutral moiety, or a therapeutic
moiety, as discussed herein. Referring now to FIG. 5, one such
particle is shown as a non-limiting example. In this figure, a
polymeric conjugate of the invention is used to form a particle 10.
The polymer forming particle 10 includes a targeting moiety 15,
present on the surface of the particle, and a biocompatible portion
17. In some cases, as shown here, targeting moiety 15 may be
conjugated to biocompatible portion 17. However, not all of
biocompatible portion 17 is shown conjugated to targeting moiety
15. For instance, in some cases, particles such as particle 10 may
be formed using a first polymer comprising biocompatible portion 17
and targeting moiety 15, and a second polymer comprising
biocompatible portion 17 but not targeting moiety 15. By
controlling the ratio of the first and second polymers, particles
having different properties may be formed, and in some cases,
libraries of such particles may be formed. In addition, contained
within the center of particle 10 is drug 12. In some cases, drug 12
may be contained within the particle due to hydrophobic effects.
For instance, the interior of the particle may be relatively
hydrophobic with respect to the surface of the particle, and the
drug may be a hydrophobic drug that associates with the relatively
hydrophobic center of the particle. As a specific example, particle
10 may contain polymers including a relatively hydrophobic
biocompatible polymer and a relatively hydrophilic targeting moiety
15, such that, during particle formation, a greater concentration
of the hydrophilic targeting moiety is exposed on the surface and a
greater concentration of the hydrophobic biocompatible polymer is
present within the interior of the particle.
[0066] Thus, various aspects of the invention are generally
directed to polymeric conjugates comprising a biocompatible
polymer, and a moiety such as a targeting moiety, an imaging
moiety, a chelating moiety, a moiety having multiple charge groups,
a neutral moiety, or a therapeutic moiety. In some cases, the
polymeric conjugate is a block copolymer, and in some embodiments,
the polymeric conjugate is amphiphilic, i.e., having a relatively
hydrophilic portion and a relatively hydrophobic portion. The
targeting moiety may be, for example, a peptide or a
polynucleotide, such as an aptamer. In some cases, the targeting
moiety is able to specifically bind to a biological substrate, for
example, a cell surface receptor. The biocompatible portion of the
polymer may be biodegradable and/or hydrolyzable, in some cases. In
some embodiments, the biocompatible polymer is a hydrophobic
polymer. Non-limiting examples of biocompatible polymers include
polylactide, polyglycolide, and/or poly(lactide-co-glycolide).
[0067] In some cases, the polymeric conjugate is part of a
controlled release system. A controlled release system, as used
herein, is a polymer combined with an active agent or a drug or
other payload, such as a therapeutic agent, a diagnostic agent, a
prognostic, a prophylactic agent, etc., and the active agent is
released from the controlled release system in a predesigned or
controlled manner. For example, the active agent may be released in
a constant manner over a predetermined period of time, the active
agent may be released in a cyclic manner over a predetermined
period of time, or an environmental condition or external event may
trigger the release of the active agent. The controlled release
polymer system may include a polymer that is biocompatible, and in
some cases, the polymer is biodegradable.
[0068] A "polymer," as used herein, is given its ordinary meaning
as used in the art, i.e., a molecular structure comprising one or
more repeat units (monomers), connected by covalent bonds. The
repeat units may all be identical, or in some cases, there may be
more than one type of repeat unit present within the polymer. In
some cases, the polymer is biologically derived, i.e., a
biopolymer. Non-limiting examples include peptides or proteins
(i.e., polymers of various amino acids), or nucleic acids such as
DNA or RNA, as discussed below. In some cases, additional moieties
may also be present in the polymer, for example biological moieties
such as those described below.
[0069] If more than one type of repeat unit is present within the
polymer, then the polymer is said to be a "copolymer." It is to be
understood that in any embodiment employing a polymer, the polymer
being employed may be a copolymer in some cases. The repeat units
forming the copolymer may be arranged in any fashion. For example,
the repeat units may be arranged in a random order, in an
alternating order, or as a "block" copolymer, i.e., comprising one
or more regions each comprising a first repeat unit (e.g., a first
block), and one or more regions each comprising a second repeat
unit (e.g., a second block), etc. Block copolymers may have two (a
diblock copolymer), three (a triblock copolymer), or more numbers
of distinct blocks.
[0070] It should be understood that, although the terms "first,"
"second," etc. may be used herein to describe various elements,
including polymeric components, these terms should not be construed
as being limiting (e.g., describing a particular order or number of
elements), but rather, as being merely descriptive, i.e., labels
that distinguish one element from another, as is commonly used
within the field of patent law. Thus, for example, although one
embodiment of the invention may be described as having a "first"
element present and a "second" element present, other embodiments
of the invention may have a "first" element present but no "second"
element present, a "second" element present but no "first" element
present, two (or more) "first" elements present, and/or two (or
more) "second" elements present, etc., and/or additional elements
such as a "first" element, a "second" element, and a "third"
element, without departing from the scope of the present
invention.
[0071] Various embodiments of the present invention are directed to
polymeric conjugates. As used herein, a "polymeric conjugate"
describes two or more polymers (such as those described herein)
that have been associated with each other, usually by covalent
bonding of the two or more polymers together. Thus, a polymeric
conjugate may comprise a first polymer and a second polymer, which
have been conjugated together to form a block copolymer where the
first polymer is a first block of the block copolymer and the
second polymer is a second block of the block copolymer. Of course,
those of ordinary skill in the art will understand that a block
copolymer may, in some cases, contain multiple blocks of polymer,
and that a "block copolymer," as used herein, is not limited to
only block copolymers having only a single first block and a single
second block. For instance, a block copolymer may comprise a first
block comprising a first polymer, a second block comprising a
second polymer, and a third block comprising a third polymer or the
first polymer, etc. In some cases, block copolymers can contain any
number of first blocks of a first polymer and second blocks of a
second polymer (and in certain cases, third blocks, fourth blocks,
etc.). In addition, it should be noted that block copolymers can
also be formed, in some instances, from other block copolymers. For
example, a first block copolymer may be conjugated to another
polymer (which may be a homopolymer, a biopolymer, another block
copolymer, etc.), to form a new block copolymer containing multiple
types of blocks, and/or to other moieties (e.g., to non-polymeric
moieties).
[0072] In some embodiments, the polymeric conjugate is amphiphilic,
i.e., having a hydrophilic portion and a hydrophobic portion, or a
relatively hydrophilic portion and a relatively hydrophobic
portion. A hydrophilic polymer is one generally that attracts water
and a hydrophobic polymer is one that generally repels water. A
hydrophilic or a hydrophobic polymer can be identified, for
example, by preparing a sample of the polymer and measuring its
contact angle with water (typically, the polymer will have a
contact angle of less than 60.degree., while a hydrophobic polymer
will have a contact angle of greater than about 60.degree.). In
some cases, the hydrophilicity of two or more polymers may be
measured relative to each other, i.e., a first polymer may be more
hydrophilic than a second polymer. For instance, the first polymer
may have a smaller contact angle than the second polymer.
[0073] In one set of embodiments, a polymeric conjugate of the
present invention includes a biocompatible polymer, i.e., the
polymer that does not typically induce an adverse response when
inserted or injected into a living subject, for example, without
significant inflammation and/or acute rejection of the polymer by
the immune system, for instance, via a T-cell response. It will be
recognized, of course, that "biocompatibility" is a relative term,
and some degree of immune response is to be expected even for
polymers that are highly compatible with living tissue. However, as
used herein, "biocompatibility" refers to the acute rejection of
material by at least a portion of the immune system, i.e., a
non-biocompatible material implanted into a subject provokes an
immune response in the subject that is severe enough such that the
rejection of the material by the immune system cannot be adequately
controlled, and often is of a degree such that the material must be
removed from the subject. One simple test to determine
biocompatibility is to expose a polymer to cells in vitro;
biocompatible polymers are polymers that typically will not result
in significant cell death at moderate concentrations, e.g., at
concentrations of 50 micrograms/10.sup.6 cells. For instance, a
biocompatible polymer may cause less than about 20% cell death when
exposed to cells such as fibroblasts or epithelial cells, even if
phagocytosed or otherwise uptaken by such cells. Non-limiting
examples of biocompatible polymers that may be useful in various
embodiments of the present invention include polydioxanone (PDO),
polyhydroxyalkanoate, polyhydroxybutyrate, poly(glycerol sebacate),
polyglycolide, polylactide, polycaprolactone, or copolymers or
derivatives including these and/or other polymers.
[0074] In certain embodiments, the biocompatible polymer is
biodegradable, i.e., the polymer is able to degrade, chemically
and/or biologically, within a physiological environment, such as
within the body. For instance, the polymer may be one that
hydrolyzes spontaneously upon exposure to water (e.g., within a
subject), the polymer may degrade upon exposure to heat (e.g., at
temperatures of about 37.degree. C.). Degradation of a polymer may
occur at varying rates, depending on the polymer or copolymer used.
For example, the half-life of the polymer (the time at which 50% of
the polymer is degraded into monomers and/or other nonpolymeric
moieties) may be on the order of days, weeks, months, or years,
depending on the polymer. The polymers may be biologically
degraded, e.g., by enzymatic activity or cellular machinery, in
some cases, for example, through exposure to a lysozyme (e.g.,
having relatively low pH). In some cases, the polymers may be
broken down into monomers and/or other nonpolymeric moieties that
cells can either reuse or dispose of without significant toxic
effect on the cells (for example, polylactide may be hydrolyzed to
form lactic acid, polyglycolide may be hydrolyzed to form glycolic
acid, etc.). Examples of biodegradable polymers include, but are
not limited to, poly(lactide) (or poly(lactic acid)),
poly(glycolide) (or poly(glycolic acid)), poly(orthoesters),
poly(caprolactones), polylysine, poly(ethylene imine), poly(acrylic
acid), poly(urethanes), poly(anhydrides), poly(esters),
poly(trimethylene carbonate), poly(ethyleneimine), poly(acrylic
acid), poly(urethane), poly(beta amino esters) or the like, and
copolymers or derivatives of these and/or other polymers, for
example, poly(lactide-co-glycolide) (PLGA).
[0075] In another set of embodiments, a polymeric conjugate of the
present invention includes a polymer able to control
immunogenicity, for example a poly(alkylene glycol) (also known as
poly(alkylene oxide)), such as poly(propylene glycol), or
poly(ethylene oxide), also known as poly(ethylene glycol) ("PEG"),
having the formula --(CH.sub.2--CH.sub.2--O).sub.n--, where n is
any positive integer. The poly(ethylene glycol) units may be
present within the polymeric conjugate in any suitable form. For
instance, the polymeric conjugate may be a block copolymer where
one of the blocks is poly(ethylene glycol). A polymeric conjugate
containing poly(ethylene glycol) repeat units is also referred to
as a "PEGylated" polymer. Such polymers can control inflammation
and/or immunogenicity (i.e., the ability to provoke an immune
response), due to the presence of the poly(ethylene glycol)
groups.
[0076] PEGylation may also be used, in some cases, to decrease
charge interaction between a polymer and a biological moiety, e.g.,
by creating a hydrophilic layer on the surface of the polymer,
which may shield the polymer from interacting with the biological
moiety. In some cases, the addition of poly(ethylene glycol) repeat
units may increase plasma half-life of the polymeric conjugate, for
instance, by decreasing the uptake of the polymeric conjugate by
the phagocytic system while decreasing transfection/uptake
efficiency by cells. Those of ordinary skill in the art will know
of methods and techniques for PEGylating a polymer, for example, by
using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride) and NHS (N-hydroxysuccinimide) to react a polymer to
a PEG group terminating in an amine, as discussed in the examples
below, by ring opening polymerization techniques (ROMP), or the
like.
[0077] In addition, certain embodiments of the invention are
directed towards copolymers containing poly(ester-ether)s, e.g.,
polymers having repeat units joined by ester bonds (e.g.,
R--C(O)--O--R' bonds) and ether bonds (e.g., R--O--R' bonds). In
some embodiments of the invention, a biodegradable polymer, such as
a hydrolyzable polymer, containing carboxylic acid groups, may be
conjugated with poly(ethylene glycol) repeat units to form a
poly(ester-ether).
[0078] In yet another set of embodiments a polymeric conjugate of
the present invention includes a targeting moiety, i.e., a moiety
able to bind to or otherwise associate with a biological entity,
for example, a membrane component, a cell surface receptor,
prostate specific membrane antigen, or the like. The term
"binding," as used herein, refers to the interaction between a
corresponding pair of molecules or portions thereof that exhibit
mutual affinity or binding capacity, typically due to specific or
non-specific binding or interaction, including, but not limited to,
biochemical, physiological, and/or chemical interactions.
"Biological binding" defines a type of interaction that occurs
between pairs of molecules including proteins, nucleic acids,
glycoproteins, carbohydrates, hormones, or the like. The term
"binding partner" refers to a molecule that can undergo binding
with a particular molecule. "Specific binding" refers to molecules,
such as polynucleotides, that are able to bind to or recognize a
binding partner (or a limited number of binding partners) to a
substantially higher degree than to other, similar biological
entities. In one set of embodiments, the targeting moiety has a
specificity (as measured via a disassociation constant) of less
than about 1 micromolar, at least about 10 micromolar, or at least
about 100 micromolar.
[0079] Non-limiting examples of biological moieties include a
peptide, a protein, an enzyme, a nucleic acid, a fatty acid, a
hormone, an antibody, a carbohydrate, a peptidoglycan, a
glycopeptide, or the like. These and other biological moieties are
discussed in detail below. In some cases, the biological moiety may
be relatively large, for example, for peptides, nucleic acids, or
the like. For example, the biological moiety may have a molecular
weight of at least about 1,000 Da, at least about 2,500 Da, at
least about 3000 Da, at least about 4000 Da, or at least about
5,000 Da, etc. Relatively large targeting moieties may be useful,
in some cases, for differentiating between cells. For instance, in
some cases, smaller targeting moieties (e.g., less than about 1000
Da) may not have adequate specificity for certain targeting
applications, such as targeting applications. In contrast, larger
molecular weight targeting moieties can offer a much higher
targeting affinity and/or specificity. For example, a targeting
moiety may offer smaller dissociation constants, e.g., tighter
binding. However, in other embodiments, the targeting moiety may be
relatively small, for example, having a molecular weight of less
than about 1,000 Da or less than about 500 Da.
[0080] In one embodiment, the targeting moiety includes a protein
or a peptide. "Proteins" and "peptides" are well-known terms in the
art, and are not precisely defined in the art in terms of the
number of amino acids that each includes. As used herein, these
terms are given their ordinary meaning in the art. Generally,
peptides are amino acid sequences of less than about 100 amino
acids in length, but can include sequences of up to 300 amino
acids. Proteins generally are considered to be molecules of at
least 100 amino acids. A protein may be, for example, a protein
drug, an antibody, an antibody fragment, a recombinant antibody, a
recombinant protein, an enzyme, or the like. In some cases, one or
more of the amino acids of the protein or peptide may be modified
in some instances, for example, by the addition of a chemical
entity such as a carbohydrate group, a phosphate group, a farnesyl
group, an isofarnesyl group, a fatty acid group, a linker for
conjugation, functionalization, or other modification, etc.
[0081] Other examples of peptides or proteins include, but are not
limited to, ankyrins, arrestins, bacterial membrane proteins,
clathrin, connexins, dystrophin, endothelin receptor, spectrin,
selectin, cytokines; chemokines; growth factors, insulin,
erythropoietin (EPO), tumor necrosis factor (TNF), neuropeptides,
neuropeptide Y, neurotensin, transforming growth factor alpha,
transforming growth factor beta, interferon (IFN), and hormones,
growth inhibitors, e.g., genistein, steroids etc; glycoproteins,
e.g., ABC transporters, platelet glycoproteins, GPIb-IX complex,
GPIIb-IIIa complex, vitronectin, thrombomodulin, CD4, CD55, CD58,
CD59, CD44, lymphocye function-associated antigen, intercellular
adhesion molecule, vascular cell adhesion molecule, Thy-1,
antiporters, CA-15-3 antigen, fibronectins, laminin,
myelin-associated glycoprotein, GAP, GAP-43.
[0082] As used herein, an "antibody" refers to a protein or
glycoprotein consisting of one or more polypeptides substantially
encoded by immunoglobulin genes or fragments of immunoglobulin
genes. The recognized immunoglobulin genes include the kappa,
lambda, alpha, gamma, delta, epsilon, and mu constant region genes,
as well as myriad immunoglobulin variable region genes. Light
chains are classified as either kappa or lambda. Heavy chains are
classified as gamma, mu, alpha, delta, or epsilon, which in turn
define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE,
respectively. A typical immunoglobulin (antibody) structural unit
is known to comprise a tetramer. Each tetramer is composed of two
identical pairs of polypeptide chains, each pair having one "light"
(about 25 kD) and one "heavy" chain (about 50-70 kD). The
N-terminus of each chain defines a variable region of about 100 to
110 or more amino acids primarily responsible for antigen
recognition. The terms variable light chain (VL) and variable heavy
chain (VH) refer to these light and heavy chains respectively.
Antibodies exist as intact immunoglobulins or as a number of well
characterized fragments produced by digestion with various
peptidases.
[0083] Non-limiting examples of antibodies and other suitable
targeting moieties include anti-cluster of differentiation antigen
CD-1 through CD-166 and the ligands or counter receptors for these
molecules; anti-cytokine antibodies, e.g., anti-IL-1 through
anti-IL-18 and the receptors for these molecules; anti-immune
receptor antibodies, antibodies against T cell receptors, major
histocompatibility complexes I and II, B cell receptors, selectin
killer inhibitory receptors, killer activating receptors, OX-40,
MadCAM-1, Gly-CAM1, integrins, cadherens, sialoadherens, Fas,
CTLA-4, Fc-gamma receptor, Fc-alpha receptors, Fc-epsilon
receptors, Fc-mu receptors, and their ligands;
anti-metalloproteinase antibodies, e.g., collagenase, MMP-1 through
MMP-8, TIMP-1, TIMP-2; anti-cell lysis/proinflammatory molecules,
e.g., perforin, complement components, prostanoids, nitrous oxide,
thromboxanes; or anti-adhesion molecules, e.g., carcioembryonic
antigens, lamins, or fibronectins.
[0084] Other examples of targeting moieties include cytokines or
cytokine receptors, such as Interleukin-1 (IL-1), IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL11, IL-12, IL-13, IL-14,
IL-15, IL-16, IL-17, IL-18, IL-1 receptor, IL-2 receptor, IL-3
receptor, IL-4 receptor, IL-5 receptor, IL-6 receptor, IL-7
receptor, IL-8 receptor, IL-9 receptor, IL-10 receptor, IL-11
receptor, IL-12 receptor, IL-13 receptor, IL-14 receptor, IL-15
receptor, IL-16 receptor, IL-17 receptor, IL-18 receptor,
lymphokine inhibitory factor, macrophage colony stimulating factor,
platelet derived growth factor, stem cell factor, tumor growth
factor beta, tumor necrosis factor, lymphotoxin, Fas, granulocyte
colony stimulating factor, granulocyte macrophage colony
stimulating factor, interferon alpha, interferon beta, interferon
gamma.
[0085] Still other examples of targeting moieties include growth
factors and protein hormones, for example, erythropoietin,
angiogenin, hepatocyte growth factor, fibroblast growth factor,
keratinocyte growth factor, nerve growth factor, tumor growth
factor alpha, thrombopoietin, thyroid stimulating factor, thyroid
releasing hormone, neurotrophin, epidermal growth factor, VEGF,
ciliary neurotrophic factor, LDL, somatomedin, insulin growth
factor, or insulin-like growth factor I and II.
[0086] Additional examples of targeting moieties include
chemokines, for example, ENA-78, ELC, GRO-alpha, GRO-beta,
GRO-gamma, HRG, LIF, IP-10, MCP-1, MCP-2, MCP-3, MCP-4, MIP-1
alpha, MIP-1 beta, MIG, MDC, NT-3, NT-4, SCF, LIF, leptin, RANTES,
lymphotactin, eotaxin-1, eotaxin-2, TARC, TECK, WAP-1, WAP-2,
GCP-1, GCP-2, alpha-chemokine receptors such as CXCR1, CXCR2,
CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, or beta-chemokine receptors such
as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, or CCR7.
[0087] In another embodiment, the targeting moiety includes a
nucleic acid. The term "nucleic acids," or "oligonucleotides," as
used herein, refers to a polymer of nucleotides. As used herein, a
"nucleotide" is given its ordinary meaning as used in the art,
i.e., a molecule comprising a sugar moiety, a phosphate group, and
a base (usually nitrogenous). Typically, the nucleotide comprises
one or more bases connected to a sugar-phosphate backbone (a base
connected only to a sugar moiety, without the phosphate group, is a
"nucleoside"). The sugars within the nucleotide may be, for
example, ribose sugars (a "ribonucleic acid," or "RNA"), or
deoxyribose sugars (a "deoxyribonucleic acid," or "DNA"). In some
cases, the polymer may comprise both ribose and deoxyribose sugars.
Examples of bases include, but not limited to, the
naturally-occurring bases (e.g., adenosine or "A," thymidine or
"T," guanosine or "G," cytidine or "C," or uridine or "U"). In some
cases, the polymer may also comprise nucleoside analogs (e.g.,
aracytidine, inosine, isoguanosine, nebularine, pseudouridine,
2,6-diaminopurine, 2-aminopurine, 2-thiothymidine,
3-deaza-5-azacytidine, 2'-deoxyuridine, 3-nitorpyrrole,
4-methylindole, 4-thiouridine, 4-thiothymidine, 2-aminoadenosine,
2-thiothymidine, 2-thiouridine, 5-bromocytidine, 5-iodouridine,
inosine, 6-azauridine, 6-chloropurine, 7-deazaadenosine,
7-deazaguanosine, 8-azaadenosine, 8-azidoadenosine, benzimidazole,
M1-methyladenosine, pyrrolo-pyrimidine, 2-amino-6-chloropurine,
3-methyl adenosine, 5-propynylcytidine, 5-propynyluridine,
5-bromouridine, 5-fluorouridine, 5-methylcytidine,
7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,
O(6)-methylguanine, 2-thiocytidine, etc.), chemically or
biologically modified bases (e.g., methylated bases), intercalated
bases, modified sugars (e.g., 2'-fluororibose, 2'-aminoribose,
2'-azidoribose, 2'-O-methylribose, L-enantiomeric nucleosides
arabinose, hexose, etc.), modified phosphate moieties (e.g.,
phosphorothioates or 5'-N-phosphoramidite linkages), and/or other
naturally and non-naturally occurring bases substitutable into the
polymer, including substituted and unsubstituted aromatic moieties.
Other suitable base and/or polymer modifications are well-known to
those of skill in the art. In some cases, the polynucleotide may
include DNA, RNA, modified DNA, modified RNA, antisense
oligonucleotides, expression plasmid systems, nucleotides, modified
nucleotides, nucleosides, modified nucleosides, aptamers, intact
genes, or combinations thereof. Other examples of polynucleotides
include interfering RNA, natural or unnatural siRNAs, shRNAs,
microRNAs, ribozymes, DNA plasmids, aptamers, antisense
oligonucleotides, randomized oligonucleotides, or ribozymes.
[0088] As a specific non-limiting example, the targeting moiety may
include an aptamer, i.e. a nucleic acid able to specifically bind a
specific target molecule, such as a biological moiety. Non-limiting
examples of aptamers include RNA aptamers and DNA aptamers. For
example, the size of the aptamer may be at least about 5 kDa, at
least about 10 kDa, at least about 15 kDa, or at least about 20
kDa. Anon-limiting example of a particular aptamer is prostate
specific membrane antigen (PSMA) aptamer (FIG. 6). The PSMA aptamer
may have the sequence TABLE-US-00001 (SEQ ID NO: 1)
GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCUUGUCAAUCCUC AUCGGC.
[0089] In still another set of embodiments a polymeric conjugate of
the present invention includes an imaging moiety or a sensing
moiety, i.e., a moiety that can be determined in some fashion,
either directly or indirectly. For instance, the imaging entity may
be fluorescent, radioactive, electron-dense, a member of a binding
pair, a substrate for an enzymatic reaction, an antigen for an
antibody, etc. In some cases, the imaging entity itself is not
directly determined, but instead interacts with a second entity in
order to effect determination; for example, coupling of the second
entity to the imaging entity may result in a determinable signal.
Non-limiting examples of imaging moieties includes fluorescent
compounds such as FITC or a FITC derivative, fluorescein, GFP, etc;
a radioactive atom, for example, .sup.3H, .sup.14C, .sup.33P,
.sup.32P, .sup.125I, .sup.131I, .sup.35S, etc.; or a heavy metal
species, for example, gold or osmium. As a specific example, an
imaging moiety may be a gold nanoparticle.
[0090] In still another set of embodiments, a polymer conjugate of
the present invention includes a therapeutic moiety, i.e., a moiety
that has a therapeutic or prophylactic effect when given to a
subject. Examples of therapeutic moieties include, but are not
limited to, antimicrobial agents, analgesics, antiinflammatory
agents, counterirritants, coagulation modifying agents, diuretics,
sympathomimetics, anorexics, antacids and other gastrointestinal
agents; antiparasitics, antidepressants, antihypertensives,
anticholinergics, stimulants, antihormones, central and respiratory
stimulants, drug antagonists, lipid-regulating agents, uricosurics,
cardiac glycosides, electrolytes, ergot and derivatives thereof,
expectorants, hypnotics and sedatives, antidiabetic agents,
dopaminergic agents, antiemetics, muscle relaxants,
para-sympathomimetics, anticonvulsants, antihistamines,
beta-blockers, purgatives, antiarrhythmics, contrast materials,
radiopharmaceuticals, antiallergic agents, tranquilizers,
vasodilators, antiviral agents, and antineoplastic or cytostatic
agents or other agents with anticancer properties, or a combination
thereof. Other suitable therapeutic moieties include contraceptives
and vitamins as well as micro- and macronutrients. Still other
examples include antiinfectives such as antibiotics and antiviral
agents; analgesics and analgesic combinations; anorexics;
antiheimintics; antiarthritics; antiasthmatic agents;
anticonvulsants; antidepressants; antidiuretic agents;
antidiarrleals; antihistamines; antiinflammatory agents;
antimigraine preparations; antinauseants; antineoplastics;
antiparkinsonism drugs; antipruritics; antipsychotics;
antipyretics, antispasmodics; anticholinergics; sympathomimetics;
xanthine derivatives; cardiovascular preparations including calcium
channel blockers and beta-blockers such as pindolol and
antiarrhythmics; antihypertensives; diuretics; vasodilators
including general coronary, peripheral and cerebral; central
nervous system stimulants; cough and cold preparations, including
decongestants; hormones such as estradiol and other steroids,
including corticosteroids; hypnotics; immunosuppressives; muscle
relaxants; parasympatholytics; psychostimulants; sedatives; and
tranquilizers; and naturally derived or genetically engineered
proteins, polysaccharides, glycoproteins, or lipoproteins.
[0091] In yet another set of embodiments a polymeric conjugate of
the present invention includes a chelating moiety, i.e., a moiety
that can bind one or more ions, typically divalent (or higher) ions
such as Ca.sup.2+, Mg.sup.2+, or Fe.sup.2+. An example of such a
moiety is ethylenediamine tetraacetic acid. In another set of
embodiments a polymeric conjugate of the present invention includes
a moiety having multiple charge groups, e.g., under physiological
conditions.
[0092] A polymeric conjugate of the present invention may be formed
using any suitable conjugation technique. For instance, two
polymers such as a targeting moiety and a biocompatible polymer, a
biocompatible polymer and a poly(ethylene glycol), etc., may be
conjugated together using techniques such as EDC-NHS chemistry
(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and
N-hydroxysuccinimide) or a reaction involving a maleimide or a
carboxylic acid, which can be conjugated to one end of a thiol, an
amine, or a similarly functionalized polyether. The conjugation of
such polymers, for instance, the conjugation of a poly(ester) and a
poly(ether) to form a poly(ester-ether), can be performed in an
organic solvent, such as, but not limited to, dichloromethane,
acetonitrile, chloroform, dimethylformamide, tetrahydrofuran,
acetone, or the like. Specific reaction conditions can be
determined by those of ordinary skill in the art using no more than
routine experimentation.
[0093] In another set of embodiments, a conjugation reaction may be
performed by reacting a polymer that comprises a carboxylic acid
functional group (e.g., a poly(ester-ether) compound) with a
polymer or other moiety (such as a targeting moiety) comprising an
amine. For instance, a targeting moiety, such as an aptamer, may be
reacted with an amine to form an amine-containing moiety, which can
then be conjugated to the carboxylic acid of the polymer. Such a
reaction may occur as a single-step reaction, i.e., the conjugation
is performed without using intermediates such as
N-hydroxysuccinimide or a maleimide. The conjugation reaction
between the amine-containing moiety and the carboxylic
acid-terminated polymer (such as a poly(ester-ether) compound) may
be achieved, in one set of embodiments, by adding the
amine-containing moiety, solubilized in an organic solvent such as
(but not limited to) dichloromethane, acetonitrile, chloroform,
tetrahydrofuran, acetone, formamide, dimethylformamide, pyridines,
dioxane, or dimethysulfoxide, to a solution containing the
carboxylic acid-terminated polymer. The carboxylic acid-terminated
polymer may be contained within an organic solvent such as, but not
limited to, dichloromethane, acetonitrile, chloroform,
dimethylformamide, tetrahydrofuran, or acetone. Reaction between
the amine-containing moiety and the carboxylic acid-terminated
polymer may occur spontaneously, in some cases. Unconjugated
macromers may be washed away after such reactions, and the polymer
may be precipitated in solvents such as, for instance, ethyl ether,
hexane, methanol, or ethanol.
[0094] As a specific example, a nuclease-stable oligonucleotide,
for instance, prostate specific membrane antigen (PSMA) aptamer,
may be prepared as a targeting moiety in a particle as follows.
Carboxylic acid modified poly(lactide-co-glycolide) (PLGA-COOH) may
be conjugated to an amine-modified heterobifunctional poly(ethylene
glycol) (NH.sub.2--PEG-COOH) to form a copolymer of PLGA-PEG-COOH.
By using an amine-modified PSMA aptamer (NH.sub.2-Apt), a triblock
polymer of PLGA-PEG-Apt may be formed by conjugating the carboxylic
acid end of the PEG to the amine functional group on the aptamer.
The multiblock polymer can then be used, for instance, as discussed
below, e.g., for therapeutic, imaging, and/or diagnostic
applications.
[0095] Another aspect of the invention is directed to particles
that include polymer conjugates such as the ones described above.
The particles may have a substantially spherical (i.e., the
particles generally appear to be spherical), or non-spherical
configuration. For instance, the particles, upon swelling or
shrinkage, may adopt a non-spherical configuration. In some cases,
the particles may include polymeric blends. For instance, a polymer
blend may be formed that includes a first polymer comprising a
targeting moiety and a biocompatible polymer, and a second polymer
comprising a biocompatible polymer but not comprising the targeting
moiety. By controlling the ratio of the first and second polymers
in the final polymer, the concentration and location of targeting
moiety in the final polymer may be readily controlled to any
suitable degree. Thus, in certain embodiments, a library of such
particles may be created, as discussed below.
[0096] In some cases, the particle is a nanoparticle, i.e., the
particle has a characteristic dimension of less than about 1
micrometer, where the characteristic dimension of a particle is the
diameter of a perfect sphere having the same volume as the
particle. For example, the particle may have a characteristic
dimension of the particle may be less than about 300 nm, less than
about 200 nm, less than about 150 nm, less than about 100 nm, less
than about 50 nm, less than about 30 nm, less than about 10 nm,
less than about 3 nm, or less than about 1 nm in some cases.
[0097] In some cases, a population of particles may be present. For
example, a population of particles may include at least 20
particles, at least 50 particles, at least 100 particles, at least
300 particles, at least 1,000 particles, at least 3,000 particles,
or at least 10,000 particles. Various embodiments of the present
invention are directed to such populations of particles. For
instance, in some embodiments, the particles may each be
substantially the same shape and/or size ("monodisperse"). For
example, the particles may have a distribution of characteristic
dimensions such that no more than about 5% or about 10% of the
particles have a characteristic dimension greater than about 10%
greater than the average characteristic dimension of the particles,
and in some cases, such that no more than about 8%, about 5%, about
3%, about 1%, about 0.3%, about 0.1%, about 0.03%, or about 0.01%
have a characteristic dimension greater than about 10% greater than
the average characteristic dimension of the particles. In some
cases, no more than about 5% of the particles have a characteristic
dimension greater than about 5%, about 3%, about 1%, about 0.3%,
about 0.1%, about 0.03%, or about 0.01% greater than the average
characteristic dimension of the particles.
[0098] In one set of embodiments, the particles may have an
interior and a surface, where the surface has a composition
different from the interior, i.e., there may be at least one
compound present in the interior but not present on the surface (or
vice versa), and/or at least one compound is present in the
interior and on the surface at differing concentrations. For
example, in one embodiment, a compound, such as a targeting moiety
(e.g., an aptamer) of a polymeric conjugate of the present
invention, may be present in both the interior and the surface of
the particle, but at a higher concentration on the surface than in
the interior of the particle, although in some cases, the
concentration in the interior of the particle may be essentially
nonzero, i.e., there is a detectable amount of the compound present
in the interior of the particle.
[0099] In some cases, the interior of the particle is more
hydrophobic than the surface of the particle. For instance, the
interior of the particle may be relatively hydrophobic with respect
to the surface of the particle, and a drug or other payload may be
hydrophobic, and readily associates with the relatively hydrophobic
center of the particle. The drug or other payload may thus be
contained within the interior of the particle, which may thus
shelter it from the external environment surrounding the particle
(or vice versa). For instance, a drug or other payload contained
within a particle administered to a subject will be protected from
a subject's body, and the body will also be isolated from the drug.
A targeting moiety present on the surface of the particle may allow
the particle to become localized at a particular targeting site,
for instance, a tumor, a disease site, a tissue, an organ, a type
of cell, etc. The drug or other payload may then, in some cases, be
released from the particle and allowed to interact locally with the
particular targeting site.
[0100] Yet another aspect of the invention is directed to polymer
particles having more than one polymer or macromolecule present,
and libraries involving such polymers or macromolecules. For
example, in one set of embodiments, particles may contain more than
one distinguishable macromolecule, and the ratios of the two (or
more) macromolecules may be independently controlled, which allows
for the control of properties of the particle. For instance, a
first macromolecule may be a polymeric conjugate comprising a
targeting moiety and a biocompatible portion, and a second
macromolecule may comprise a biocompatible portion but not contain
the targeting moiety, or the second macromolecule may contain a
distinguishable biocompatible portion from the first macromolecule.
Control of the amounts of these macromolecules within the polymeric
particle may thus be used to control various physical, biological,
or chemical properties of the particle, for instance, the size of
the particle (e.g., by varying the molecular weights of one or both
polymers), the surface charge (e.g., by controlling the ratios of
the polymers if the polymers have different charges or terminal
groups), the surface hydrophilicity (e.g., if the polymers have
different molecular weights and/or hydrophilicities), the surface
density of the targeting moiety (e.g., by controlling the ratios of
the two or more polymers), etc.
[0101] As a specific example, a particle may comprise a first
macromolecule comprising a poly(ethylene glycol) and a targeting
moiety conjugated to the poly(ethylene glycol), and a second
macromolecule comprising the poly(ethylene glycol) but not the
targeting moiety, or comprising both the poly(ethylene glycol) and
the targeting moiety, where the poly(ethylene glycol) of the second
macromolecule has a different length (or number of repeat units)
than the poly(ethylene glycol) of the first macromolecule. As
another example, a particle may comprise a first macromolecule
comprising a first biocompatible portion and a targeting moiety,
and a second macromolecule comprising a second biocompatible
portion different from the first biocompatible portion (e.g.,
having a different composition, a substantially different number of
repeat units, etc.) and the targeting moiety. As yet another
example, a first macromolecule may comprise a biocompatible portion
and a first targeting moiety, and a second macromolecule may
comprise a biocompatible portion and a second targeting moiety
different from the first targeting moiety.
[0102] Libraries of such particles may also be formed. For example,
by varying the ratios of the two (or more) polymers within the
particle, libraries of particles may be formed, which may be
useful, for example, for screening tests, high-throughput assays,
or the like. Entities within the library may vary by properties
such as those described above, and in some cases, more than one
property of the particles may be varied within the library.
Accordingly, one embodiment of the invention is directed to a
library of nanoparticles having different ratios of polymers with
differing properties. The library may include any suitable ratio(s)
of the polymers or macromolecules. For example, in a particle
having a first macromolecule and a second macromolecule, the first
and second macromolecule may be present in a ratio of 0 to about
5%:1, about 10%:1, about 15%:1, about 20%:1, about 5%1 to about
10%:1, etc.
[0103] Another aspect of the present invention is directed to a
"payload," or a species (or more than one species) contained within
a particle, such as those described above. For instance, the
targeting moiety may target or cause the particle to become
localized at specific portions within a subject, and the payload
may be delivered to those portions. For example, a targeting
portion may cause the particles to become localized to a tumor, a
disease site, a tissue, an organ, a type of cell, etc. within the
body of a subject, depending on the targeting moiety used. The
subject may be a human or non-human animal. Examples of subjects
include, but are not limited to, a mammal such as a dog, a cat, a
horse, a donkey, a rabbit, a cow, a pig, a sheep, a goat, a rat, a
mouse, a guinea pig, a hamster, a primate, or the like.
[0104] Those of ordinary skill in the art will be identify
targeting species specific to a targeting moiety of interest, as
previously discussed; for example, PSMA aptamer may become
localized to prostate cancer cells. Other examples of payloads
include, but are not limited to, nucleic acids such as DNA or RNA
(e.g., for RNA interference), peptides or proteins, enzymes,
antibodies, carbohydrates, small molecules (e.g., having a
molecular weight of less than about 1000 Da), or the like.
[0105] In one set of embodiments, the payload is a drug or a
combination of more than one drug. Such particles may be useful,
for example, in embodiments where a targeting moiety may be used to
direct a particle containing a drug to a particular localized
location within a subject, e.g., to allow localized delivery of the
drug to occur. Non-limiting examples of potentially suitable drugs
include antimicrobial agents, analgesics, antiinflammatory agents,
counterirritants, coagulation modifying agents, diuretics,
sympathomimetics, anorexics, antacids and other gastrointestinal
agents; antiparasitics, antidepressants, antihypertensives,
anticholinergics, stimulants, antihormones, central and respiratory
stimulants, drug antagonists, lipid-regulating agents, uricosurics,
cardiac glycosides, electrolytes, ergot and derivatives thereof,
expectorants, hypnotics and sedatives, antidiabetic agents,
dopaminergic agents, antiemetics, muscle relaxants,
para-sympathomimetics, anticonvulsants, antihistamines,
beta-blockers, purgatives, antiarrhythmics, contrast materials,
radiopharmaceuticals, antiallergic agents, tranquilizers,
vasodilators, antiviral agents, and antineoplastic or cytostatic
agents or other agents with anticancer properties, or a combination
thereof. Other suitable medicaments may be selected from
contraceptives and vitamins as well as micro- and macronutrients.
Still other examples include antiinfectives such as antibiotics and
antiviral agents; analgesics and analgesic combinations; anorexics;
antiheimintics; antiarthritics; antiasthmatic agents;
anticonvulsants; antidepressants; antidiuretic agents;
antidiarrleals; antihistamines; antiinflammatory agents;
antimigraine preparations; antinauseants; antineoplastics;
antiparkinsonism drugs; antipruritics; antipsychotics;
antipyretics, antispasmodics; anticholinergics; sympathomimetics;
xanthine derivatives; cardiovascular preparations including calcium
channel blockers and beta-blockers such as pindolol and
antiarrhythmics; antihypertensives; diuretics; vasodilators
including general coronary, peripheral and cerebral; central
nervous system stimulants; cough and cold preparations, including
decongestants; hormones such as estradiol and other steroids,
including corticosteroids; hypnotics; immunosuppressives; muscle
relaxants; parasympatholytics; psychostimulants; sedatives; and
tranquilizers; and naturally derived or genetically engineered
proteins, polysaccharides, glycoproteins, or lipoproteins.
[0106] Specific non-limiting examples of drugs include doxorubicin,
mitomycin, cisplatin, daunorubicin, bleomycin, actinomycin D,
neocarzinostatin, carboplatin, stratoplatin, Ara-C. Other examples
include Capoten, Monopril, Pravachol, Avapro, Plavix, Cefzil,
Duricef/Ultracef, Azactam, Videx, Zerit, Maxipime, VePesid,
Paraplatin, Platinol, Taxol, UFT, Buspar, Serzone, Stadol NS,
Estrace, Glucophage (Bristol-Myers Squibb); Ceclor, Lorabid,
Dynabac, Prozac, Darvon, Permax, Zyprexa, Humalog, Axid, Gemzar,
Evista (Eli Lily); Vasotec/Vaseretic, Mevacor, Zocor,
Prinivil/Prinizide, Plendil, Cozaar/Hyzaar, Pepcid, Prilosec,
Primaxin, Noroxin, Recombivax HB, Varivax, Timoptic/XE, Trusopt,
Proscar, Fosamax, Sinemet, Crixivan, Propecia, Vioxx, Singulair,
Maxalt, Ivermectin (Merck & Co.); Diflucan, Unasyn, Sulperazon,
Zithromax, Trovan, Procardia XL, Cardura, Norvasc, Dofetilide,
Feldene, Zoloft, Zeldox, Glucotrol XL, Zyrtec, Eletriptan, Viagra,
Droloxifene, Aricept, Lipitor (Pfizer); Vantin, Rescriptor,
Vistide, Genotropin, Micronase/Glyn./Glyb., Fragmin, Total Medrol,
Xanax/alprazolam, Sermion, Halcion/triazolam, Freedox, Dostinex,
Edronax, Mirapex, Pharmorubicin, Adriamycin, Camptosar, Remisar,
Depo-Provera, Caverject, Detrusitol, Estring, Healon, Xalatan,
Rogaine (Pharmacia & Upjohn); Lopid, Accrupil, Dilantin,
Cognex, Neurontin, Loestrin, Dilzem, Fempatch, Estrostep, Rezulin,
Lipitor, Omnicef, FemHRT, Suramin, or Clinafloxacin (Warner
Lambert).
[0107] As another example, if the targeting moiety targets a cancer
cell, then the payload may be an anti-cancer drug such as
20-epi-1,25 dihydroxyvitamin D3,4-ipomeanol, 5-ethynyluracil,
9-dihydrotaxol, abiraterone, acivicin, aclarubicin, acodazole
hydrochloride, acronine, acylfulvene, adecypenol, adozelesin,
aldesleukin, all-tk antagonists, altretamine, ambamustine,
ambomycin, ametantrone acetate, amidox, amifostine,
aminoglutethimide, aminolevulinic acid, amrubicin, amsacrine,
anagrelide, anastrozole, andrographolide, angiogenesis inhibitors,
antagonist D, antagonist G, antarelix, anthramycin,
anti-dorsalizing morphogenetic protein-1, antiestrogen,
antineoplaston, antisense oligonucleotides, aphidicolin glycinate,
apoptosis gene modulators, apoptosis regulators, apurinic acid,
ARA-CDP-DL-PTBA, arginine deaminase, asparaginase, asperlin,
asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2,
axinastatin 3, azacitidine, azasetron, azatoxin, azatyrosine,
azetepa, azotomycin, baccatin III derivatives, balanol, batimastat,
benzochlorins, benzodepa, benzoylstaurosporine, beta lactam
derivatives, beta-alethine, betaclamycin B, betulinic acid, BFGF
inhibitor, bicalutamide, bisantrene, bisantrene hydrochloride,
bisaziridinylspermine, bisnafide, bisnafide dimesylate, bistratene
A, bizelesin, bleomycin, bleomycin sulfate, BRC/ABL antagonists,
breflate, brequinar sodium, bropirimine, budotitane, busulfan,
buthionine sulfoximine, cactinomycin, calcipotriol, calphostin C,
calusterone, camptothecin derivatives, canarypox IL-2,
capecitabine, caracemide, carbetimer, carboplatin,
carboxamide-amino-triazole, carboxyamidotriazole, carest M3,
carmustine, cam 700, cartilage derived inhibitor, carubicin
hydrochloride, carzelesin, casein kinase inhibitors,
castanospermine, cecropin B, cedefingol, cetrorelix, chlorambucil,
chlorins, chloroquinoxaline sulfonamide, cicaprost, cirolemycin,
cisplatin, cis-porphyrin, cladribine, clomifene analogs,
clotrimazole, collismycin A, collismycin B, combretastatin A4,
combretastatin analog, conagenin, crambescidin 816, crisnatol,
crisnatol mesylate, cryptophycin 8, cryptophycin A derivatives,
curacin A, cyclopentanthraquinones, cyclophosphamide, cycloplatam,
cypemycin, cytarabine, cytarabine ocfosfate, cytolytic factor,
cytostatin, dacarbazine, dacliximab, dactinomycin, daunorubicin
hydrochloride, decitabine, dehydrodidemnin B, deslorelin,
dexifosfamide, dexormaplatin, dexrazoxane, dexverapamil,
dezaguanine, dezaguanine mesylate, diaziquone, didemnin B, didox,
diethylnorspermine, dihydro-5-azacytidine, dioxamycin, diphenyl
spiromustine, docetaxel, docosanol, dolasetron, doxifluridine,
doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene
citrate, dromostanolone propionate, dronabinol, duazomycin,
duocarmycin SA, ebselen, ecomustine, edatrexate, edelfosine,
edrecolomab, eflomithine, eflomithine hydrochloride, elemene,
elsamitrucin, emitefur, enloplatin, enpromate, epipropidine,
epirubicin, epirubicin hydrochloride, epristeride, erbulozole,
erythrocyte gene therapy vector system, esorubicin hydrochloride,
estramustine, estramustine analog, estramustine phosphate sodium,
estrogen agonists, estrogen antagonists, etanidazole, etoposide,
etoposide phosphate, etoprine, exemestane, fadrozole, fadrozole
hydrochloride, fazarabine, fenretinide, filgrastim, finasteride,
flavopiridol, flezelastine, floxuridine, fluasterone, fludarabine,
fludarabine phosphate, fluorodaunorunicin hydrochloride,
fluorouracil, fluorocitabine, forfenimex, formestane, fosquidone,
fostriecin, fostriecin sodium, fotemustine, gadolinium texaphyrin,
gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors,
gemcitabine, gemcitabine hydrochloride, glutathione inhibitors,
hepsulfam, heregulin, hexamethylene bisacetamide, hydroxyurea,
hypericin, ibandronic acid, idarubicin, idarubicin hydrochloride,
idoxifene, idramantone, ifosfamide, ilmofosine, ilomastat,
imidazoacridones, imiquimod, immunostimulant peptides, insulin-like
growth factor-1 receptor inhibitor, interferon agonists, interferon
alpha-2A, interferon alpha-2B, interferon alpha-N1, interferon
alpha-N3, interferon beta-IA, interferon gamma-IB, interferons,
interleukins, iobenguane, iododoxorubicin, iproplatin, irinotecan,
irinotecan hydrochloride, iroplact, irsogladine, isobengazole,
isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F,
lamellarin-N triacetate, lanreotide, lanreotide acetate,
leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole,
leukemia inhibiting factor, leukocyte alpha interferon, leuprolide
acetate, leuprolide/estrogen/progesterone, leuprorelin, levamisole,
liarozole, liarozole hydrochloride, linear polyamine analog,
lipophilic disaccharide peptide, lipophilic platinum compounds,
lissoclinamide 7, lobaplatin, lombricine, lometrexol, lometrexol
sodium, lomustine, lonidamine, losoxantrone, losoxantrone
hydrochloride, lovastatin, loxoribine, lurtotecan, lutetium
texaphyrin, lysofylline, lytic peptides, maitansine, mannostatin A,
marimastat, masoprocol, maspin, matrilysin inhibitors, matrix
metalloproteinase inhibitors, maytansine, mechlorethamine
hydrochloride, megestrol acetate, melengestrol acetate, melphalan,
menogaril, merbarone, mercaptopurine, meterelin, methioninase,
methotrexate, methotrexate sodium, metoclopramide, metoprine,
meturedepa, microalgal protein kinase C inhibitors, MIF inhibitor,
mifepristone, miltefosine, mirimostim, mismatched double stranded
RNA, mitindomide, mitocarcin, mitocromin, mitogillin, mitoguazone,
mitolactol, mitomalcin, mitomycin, mitomycin analogs, mitonafide,
mitosper, mitotane, mitotoxin fibroblast growth factor-saporin,
mitoxantrone, mitoxantrone hydrochloride, mofarotene, molgramostim,
monoclonal antibody, human chorionic gonadotrophin, monophosphoryl
lipid a/myobacterium cell wall SK, mopidamol, multiple drug
resistance gene inhibitor, multiple tumor suppressor 1-based
therapy, mustard anticancer agent, mycaperoxide B, mycobacterial
cell wall extract, mycophenolic acid, myriaporone,
n-acetyldinaline, nafarelin, nagrestip, naloxone/pentazocine,
napavin, naphterpin, nartograstim, nedaplatin, nemorubicin,
neridronic acid, neutral endopeptidase, nilutamide, nisamycin,
nitric oxide modulators, nitroxide antioxidant, nitrullyn,
nocodazole, nogalamycin, n-substituted benzamides,
06-benzylguanine, octreotide, okicenone, oligonucleotides,
onapristone, ondansetron, oracin, oral cytokine inducer,
ormaplatin, osaterone, oxaliplatin, oxaunomycin, oxisuran,
paclitaxel, paclitaxel analogs, paclitaxel derivatives, palauamine,
palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene,
parabactin, pazelliptine, pegaspargase, peldesine, peliomycin,
pentamustine, pentosan polysulfate sodium, pentostatin, pentrozole,
peplomycin sulfate, perflubron, perfosfamide, perillyl alcohol,
phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil,
pilocarpine hydrochloride, pipobroman, piposulfan, pirarubicin,
piritrexim, piroxantrone hydrochloride, placetin A, placetin B,
plasminogen activator inhibitor, platinum complex, platinum
compounds, platinum-triamine complex, plicamycin, plomestane,
porfimer sodium, porfiromycin, prednimustine, procarbazine
hydrochloride, propyl bis-acridone, prostaglandin J2, prostatic
carcinoma antiandrogen, proteasome inhibitors, protein A-based
immune modulator, protein kinase C inhibitor, protein tyrosine
phosphatase inhibitors, purine nucleoside phosphorylase inhibitors,
puromycin, puromycin hydrochloride, purpurins, pyrazofurin,
pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene
conjugate, RAF antagonists, raltitrexed, ramosetron, RAS farnesyl
protein transferase inhibitors, RAS inhibitors, RAS-GAP inhibitor,
retelliptine demethylated, rhenium RE 186 etidronate, rhizoxin,
riboprine, ribozymes, RII retinamide, RNAi, rogletimide,
rohitukine, romurtide, roquinimex, rubiginone B1, ruboxyl,
safingol, safingol hydrochloride, saintopin, sarcnu, sarcophytol A,
sargramostim, SDI 1 mimetics, semustine, senescence derived
inhibitor 1, sense oligonucleotides, signal transduction
inhibitors, signal transduction modulators, simtrazene, single
chain antigen binding protein, sizofuran, sobuzoxane, sodium
borocaptate, sodium phenylacetate, solverol, somatomedin binding
protein, sonermin, sparfosate sodium, sparfosic acid, sparsomycin,
spicamycin D, spirogermanium hydrochloride, spiromustine,
spiroplatin, splenopentin, spongistatin 1, squalamine, stem cell
inhibitor, stem-cell division inhibitors, stipiamide,
streptonigrin, streptozocin, stromelysin inhibitors, sulfinosine,
sulofenur, superactive vasoactive intestinal peptide antagonist,
suradista, suramin, swainsonine, synthetic glycosaminoglycans,
talisomycin, tallimustine, tamoxifen methiodide, tauromustine,
tazarotene, tecogalan sodium, tegafur, tellurapyrylium, telomerase
inhibitors, teloxantrone hydrochloride, temoporfin, temozolomide,
teniposide, teroxirone, testolactone, tetrachlorodecaoxide,
tetrazomine, thaliblastine, thalidomide, thiamiprine, thiocoraline,
thioguanine, thiotepa, thrombopoietin, thrombopoietin mimetic,
thymalfasin, thymopoietin receptor agonist, thymotrinan, thyroid
stimulating hormone, tiazofurin, tin ethyl etiopurpurin,
tirapazamine, titanocene dichloride, topotecan hydrochloride,
topsentin, toremifene, toremifene citrate, totipotent stem cell
factor, translation inhibitors, trestolone acetate, tretinoin,
triacetyluridine, triciribine, triciribine phosphate, trimetrexate,
trimetrexate glucuronate, triptorelin, tropisetron, tubulozole
hydrochloride, turosteride, tyrosine kinase inhibitors,
tyrphostins, UBC inhibitors, ubenimex, uracil mustard, uredepa,
urogenital sinus-derived growth inhibitory factor, urokinase
receptor antagonists, vapreotide, variolin B, velaresol, veramine,
verdins, verteporfin, vinblastine sulfate, vincristine sulfate,
vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate
sulfate, vinleurosine sulfate, vinorelbine, vinorelbine tartrate,
vinrosidine sulfate, vinxaltine, vinzolidine sulfate, vitaxin,
vorozole, zanoterone, zeniplatin, zilascorb, zinostatin, zinostatin
stimalamer, or zorubicin hydrochloride.
[0108] Further specific non-limiting examples of drugs that can be
included within a particle of the present invention include
acebutolol, acetaminophen, acetohydroxamic acid, acetophenazine,
acyclovir, adrenocorticoids, allopurinol, alprazolam, aluminum
hydroxide, amantadine, ambenonium, amiloride, aminobenzoate
potassium, amobarbital, amoxicillin, amphetamine, ampicillin,
androgens, anesthetics, anticoagulants, anticonvulsants-dione type,
antithyroid medicine, appetite suppressants, aspirin, atenolol,
atropine, azatadine, bacampicillin, baclofen, beclomethasone,
belladonna, bendroflumethiazide, benzoyl peroxide, benzthiazide,
benztropine, betamethasone, betha nechol, biperiden, bisacodyl,
bromocriptine, bromodiphenhydramine, brompheniramine, buclizine,
bumetanide, busulfan, butabarbital, butaperazine, caffeine, calcium
carbonate, captopril, carbamazepine, carbenicillin, carbidopa &
levodopa, carbinoxamine inhibitors, carbonic anhydsase,
carisoprodol, carphenazine, cascara, cefaclor, cefadroxil,
cephalexin, cephradine, chlophedianol, chloral hydrate,
chlorambucil, chloramphenicol, chlordiazepoxide, chloroquine,
chlorothiazide, chlorotrianisene, chlorpheniramine, 6.times.
chlorpromazine, chlorpropamide, chlorprothixene, chlorthalidone,
chlorzoxazone, cholestyramine, cimetidine, cinoxacin, clemastine,
clidinium, clindamycin, clofibrate, clomiphere, clonidine,
clorazepate, cloxacillin, colochicine, coloestipol, conjugated
estrogen, contraceptives, cortisone, cromolyn, cyclacillin,
cyclandelate, cyclizine, cyclobenzaprine, cyclophosphamide,
cyclothiazide, cycrimine, cyproheptadine, danazol, danthron,
dantrolene, dapsone, dextroamphetamine, dexamethasone,
dexchlorpheniramine, dextromethorphan, diazepan, dicloxacillin,
dicyclomine, diethylstilbestrol, diflunisal, digitalis, diltiazen,
dimenhydrinate, dimethindene, diphenhydramine, diphenidol,
diphenoxylate & atrophive, diphenylopyraline, dipyradamole,
disopyramide, disulfuram, divalporex, docusate calcium, docusate
potassium, docusate sodium, doxyloamine, dronabinol ephedrine,
epinephrine, ergoloidmesylates, ergonovine, ergotamine,
erythromycins, esterified estrogens, estradiol, estrogen, estrone,
estropipute, etharynic acid, ethchlorvynol, ethinyl estradiol,
ethopropazine, ethosaximide, ethotoin, fenoprofen, ferrous
fumarate, ferrous gluconate, ferrous sulfate, flavoxate, flecamide,
fluphenazine, fluprednisolone, flurazepam, folic acid, furosemide,
gemfibrozil, glipizide, glyburide, glycopyrrolate, gold compounds,
griseofiwin, guaifenesin, guanabenz, guanadrel, guanethidine,
halazepam, haloperidol, hetacillin, hexobarbital, hydralazine,
hydrochlorothiazide, hydrocortisone (cortisol), hydroflunethiazide,
hydroxychloroquine, hydroxyzine, hyoscyamine, ibuprofen,
indapamide, indomethacin, insulin, iofoquinol, iron-polysaccharide,
isoetharine, isoniazid, isopropamide isoproterenol, isotretinoin,
isoxsuprine, kaolin & pectin, ketoconazole, lactulose,
levodopa, lincomycin liothyronine, liotrix, lithium, loperamide,
lorazepam, magnesium hydroxide, magnesium sulfate, magnesium
trisilicate, maprotiline, meclizine, meclofenamate,
medroxyproyesterone, melenamic acid, melphalan, mephenyloin,
mephobarbital, meprobamate, mercaptopurine, mesoridazine,
metaproterenol, metaxalone, methamphetamine, methaqualone,
metharbital, methenamine, methicillin, methocarbamol, methotrexate,
methsuximide, methyclothinzide, methylcellulos, methyldopa,
methylergonovine, methylphenidate, methylprednisolone,
methysergide, metoclopramide, matolazone, metoprolol,
metronidazole, minoxidil, mitotane, monamine oxidase inhibitors,
nadolol, nafcillin, nalidixic acid, naproxen, narcotic analgesics,
neomycin, neostigmine, niacin, nicotine, nifedipine, nitrates,
nitrofurantoin, nomifensine, norethindrone, norethindrone acetate,
norgestrel, nylidrin, nystafin, orphenadrine, oxacillin, oxazepam,
oxprenolol, oxymetazoline, oxyphenbutazone, pancrelipase,
pantothenic acid, papaverine, paraaminosalicylic acid,
paramethasone, paregoric, pemoline, penicillamine, penicillin,
penicillin-v, pentobarbital, perphenazine, phenacetin,
phenazopyridine, pheniramine, phenobarbital, phenolphthalein,
phenprocoumon, phensuximide, phenylbutazone, phenylephrine,
phenylpropanolamine, phenyl toloxamine, phenyloin, pilocarpine,
pindolol, piper acetazine, piroxicam, poloxamer, polycarbophil
calcium, polythiazide, potassium supplements, pruzepam, prazosin,
prednisolone, prednisone, primidone, probenecid, probucol,
procainamide, procarbazine, prochlorperazine, procyclidine,
promazine, promethazine, propantheline, propranolol,
pseudoephedrine, psoralens, syllium, pyridostigmine, pyrodoxine,
pyrilamine, pyrvinium, quinestrol, quinethazone, uinidine, quinine,
ranitidine, rauwolfia alkaloids, riboflavin, rifampin, ritodrine,
alicylates, scopolamine, secobarbital, senna, sannosides a & b,
simethicone, sodium bicarbonate, sodium phosphate, sodium fluoride,
spironolactone, sucrulfate, sulfacytine, sulfamethoxazole,
sulfasalazine, sulfinpyrazone, sulfisoxazole, sulindac, talbutal,
tamazepam, terbutaline, terfenadine, terphinhydrate, teracyclines,
thiabendazole, thiamine, thioridazine, thiothixene, thyroblobulin,
thyroid, thyroxine, ticarcillin, timolol, tocamide, tolazamide,
tolbutamide, tolmetin trozodone, tretinoin, triamcinolone,
trianterene, triazolam, trichlormethiazide, tricyclic
antidepressants, tridhexethyl, trifluoperazine, triflupromazine,
trihexyphenidyl, trimeprazine, trimethobenzamine, trimethoprim,
tripclennamine, triprolidine, valproic acid, verapamil, vitamin A,
vitamin B12, vitamin C, vitamin 1), vitamin E, vitamin K, xanthine,
and the like.
[0109] In another set of embodiments, the payload is a diagnostic
agent. For example, the payload may be a fluorescent molecule; a
gas; a metal; a commercially available imaging agents used in
positron emissions tomography (PET), computer assisted tomography
(CAT), single photon emission computerized tomography, x-ray,
fluoroscopy, and magnetic resonance imaging (MRI); or a contrast
agents. Non-limiting examples of suitable materials for use as
contrast agents in MRI include gadolinium chelates, as well as
iron, magnesium, manganese, copper, and chromium. Examples of
materials useful for CAT and x-ray imaging include, but are not
limited to, iodine-based materials.
[0110] As another example, the payload may include a radionuclide,
e.g., for use as a therapeutic, diagnostic, or prognostic agents.
Among the radionuclides used, gamma-emitters, positron-emitters,
and X-ray emitters are suitable for diagnostic and/or therapy,
while beta emitters and alpha-emitters may also be used for
therapy. Suitable radionuclides for forming use with various
embodiments of the present invention include, but are not limited
to, .sup.123I, .sup.125I, .sup.130I, .sup.131I, .sup.133I,
.sup.135I, .sup.47Sc, .sup.72As, .sup.72Sc, .sup.90Y, .sup.88Y,
.sup.97Ru, .sup.100Pd, .sup.101mRh, .sup.119Sb, .sup.128Ba,
.sup.197Hg, .sup.211At, .sup.212Bi, .sup.212Pb, .sup.109Pd,
.sup.111In, .sup.67Ga, .sup.68Ga, .sup.67Cu, .sup.75Br, .sup.77Br,
.sup.99mTc, .sup.14C, .sup.13N, .sup.15O, .sup.32P, .sup.33P, or
.sup.18F. The radionuclides may be contained within the particle
(e.g., as a separate species), and/or form part of a macromolecule
or polymer that forms the particle.
[0111] Another aspect of the invention is directed to systems and
methods of making such particles. In one set of embodiments, the
particles are formed by providing a solution comprising one or more
polymers, and contacting the solution with a polymer nonsolvent to
produce the particle. The solution may be miscible or immiscible
with the polymer nonsolvent. For example, as is discussed in the
examples, a water-miscible liquid such as acetonitrile may contain
the polymers, and particles are formed as the acetonitrile is
contacted with water, a polymer nonsolvent, e.g., by pouring the
acetonitrile into the water at a controlled rate. The polymer
contained within the solution, upon contact with the polymer
nonsolvent, may then precipitate to form particles such as
nanoparticles.
[0112] Two liquids are said to be "immiscible" or not miscible,
with each other when one is not soluble in the other to a level of
at least 10% by weight at ambient temperature and pressure.
Typically, an organic solution (e.g., dichloromethane,
acetonitrile, chloroform, tetrahydrofuran, acetone, formamide,
dimethylformamide, pyridines, dioxane, dimethysulfoxide, etc.) and
an aqueous liquid (e.g., water, or water containing dissolved salts
or other species, cell or biological media, ethanol, etc.) are
immiscible with respect to each other. For example, the first
solution may be poured into the second solution (at a suitable rate
or speed). In some cases, particles such as nanoparticles may be
formed as the first solution contacts the immiscible second liquid,
e.g., precipitation of the polymer upon contact causes the polymer
to form nanoparticles while the first solution poured into the
second liquid, and in some cases, for example, when the rate of
introduction is carefully controlled and kept at a relatively slow
rate, nanoparticles may form. The control of such particle
formation can be readily optimized by one of ordinary skill in the
art using only routine experimentation.
[0113] Once the inventive conjugates have been prepared, they may
be combined with pharmaceutical acceptable carriers to form a
pharmaceutical composition, according to another aspect of the
invention. As would be appreciated by one of skill in this art, the
carriers may be chosen based on the route of administration as
described below, the location of the target issue, the drug being
delivered, the time course of delivery of the drug, etc.
[0114] As used herein, the term "pharmaceutically acceptable
carrier" means a non-toxic, inert solid, semi-solid or liquid
filler, diluent, encapsulating material or formulation auxiliary of
any type. Remington's Pharmaceutical Sciences Ed. by Gennaro, Mack
Publishing, Easton, Pa., 1995 discloses various carriers used in
formulating pharmaceutical compositions and known techniques for
the preparation thereof. Some examples of materials which can serve
as pharmaceutically acceptable carriers include, but are not
limited to, sugars such as lactose, glucose, and sucrose; starches
such as corn starch and potato starch; cellulose and its
derivatives such as sodium carboxymethyl cellulose, ethyl
cellulose, and cellulose acetate; powdered tragacanth; malt;
gelatin; talc; excipients such as cocoa butter and suppository
waxes; oils such as peanut oil, cottonseed oil; safflower oil;
sesame oil; olive oil; corn oil and soybean oil; glycols such as
propylene glycol; esters such as ethyl oleate and ethyl laurate;
agar; detergents such as TWEEN.TM. 80; buffering agents such as
magnesium hydroxide and aluminum hydroxide; alginic acid;
pyrogen-free water; isotonic saline; Ringer's solution; ethyl
alcohol; and phosphate buffer solutions, as well as other non-toxic
compatible lubricants such as sodium lauryl sulfate and magnesium
stearate, as well as coloring agents, releasing agents, coating
agents, sweetening, flavoring and perfuming agents, preservatives
and antioxidants can also be present in the composition, according
to the judgment of the formulator. If filtration or other terminal
sterilization methods are not feasible, the formulations can be
manufactured under aseptic conditions.
[0115] The pharmaceutical compositions of this invention can be
administered to a patient by any means known in the art including
oral and parenteral routes. The term "patient," as used herein,
refers to humans as well as non-humans, including, for example,
mammals, birds, reptiles, amphibians, and fish. For instance, the
non-humans may be mammals (e.g., a rodent, a mouse, a rat, a
rabbit, a monkey, a dog, a cat, a primate, or a pig). In certain
embodiments parenteral routes are desirable since they avoid
contact with the digestive enzymes that are found in the alimentary
canal. According to such embodiments, inventive compositions may be
administered by injection (e.g., intravenous, subcutaneous or
intramuscular, intraperitoneal injection), rectally, vaginally,
topically (as by powders, creams, ointments, or drops), or by
inhalation (as by sprays).
[0116] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension, or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.,
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables. In one
embodiment, the inventive conjugate is suspended in a carrier fluid
comprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v)
TWEEN.TM. 80. The injectable formulations can be sterilized, for
example, by filtration through a bacteria-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0117] Compositions for rectal or vaginal administration may be
suppositories which can be prepared by mixing the inventive
conjugate with suitable non-irritating excipients or carriers such
as cocoa butter, polyethylene glycol, or a suppository wax which
are solid at ambient temperature but liquid at body temperature and
therefore melt in the rectum or vaginal cavity and release the
inventive conjugate.
[0118] Dosage forms for topical or transdermal administration of an
inventive pharmaceutical composition include ointments, pastes,
creams, lotions, gels, powders, solutions, sprays, inhalants, or
patches. The inventive conjugate is admixed under sterile
conditions with a pharmaceutically acceptable carrier and any
needed preservatives or buffers as may be required. Ophthalmic
formulations, ear drops, and eye drops are also contemplated as
being within the scope of this invention. The ointments, pastes,
creams, and gels may contain, in addition to the inventive
conjugates of this invention, excipients such as animal and
vegetable fats, oils, waxes, paraffins, starch, tragacanth,
cellulose derivatives, polyethylene glycols, silicones, bentonites,
silicic acid, talc, and zinc oxide, or mixtures thereof.
Transdermal patches have the added advantage of providing
controlled delivery of a compound to the body. Such dosage forms
can be made by dissolving or dispensing the inventive conjugates in
a proper medium. Absorption enhancers can also be used to increase
the flux of the compound across the skin. The rate can be
controlled by either providing a rate controlling membrane or by
dispersing the inventive conjugates in a polymer matrix or gel.
[0119] Powders and sprays can contain, in addition to the inventive
conjugates of this invention, excipients such as lactose, talc,
silicic acid, aluminum hydroxide, calcium silicates, and polyamide
powder, or mixtures thereof. Sprays can additionally contain
customary propellants such as chlorofluorohydrocarbons.
[0120] When administered orally, the inventive conjugates can be,
but are not necessarily, encapsulated. A variety of suitable
encapsulation systems are known in the art ("Microcapsules and
Nanoparticles in Medicine and Pharmacy," Edited by Doubrow, M., CRC
Press, Boca Raton, 1992; Mathiowitz and Langer J. Control. Release
5:13, 1987; Mathiowitz et al. Reactive Polymers 6:275, 1987;
Mathiowitz et al. J. Appl. Polymer Sci. 35:755, 1988; Langer Acc.
Chem. Res. 33:94, 2000; Langer J. Control. Release 62:7, 1999;
Uhrich et al. Chem. Rev. 99:3181, 1999; Zhou et al. J. Control.
Release 75:27, 2001; and Hanes et al. Pharm. Biotechnol. 6:389,
1995). The inventive conjugates may be encapsulated within
biodegradable polymeric microspheres or liposomes. Examples of
natural and synthetic polymers useful in the preparation of
biodegradable microspheres include carbohydrates such as alginate,
cellulose, polyhydroxyalkanoates, polyamides, polyphosphazenes,
polypropylfumarates, polyethers, polyacetals, polycyanoacrylates,
biodegradable polyurethanes, polycarbonates, polyanhydrides,
polyhydroxyacids, poly(ortho esters), and other biodegradable
polyesters. Examples of lipids useful in liposome production
include phosphatidyl compounds, such as phosphatidylglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingolipids, cerebrosides, and gangliosides.
[0121] Pharmaceutical compositions for oral administration can be
liquid or solid. Liquid dosage forms suitable for oral
administration of inventive compositions include pharmaceutically
acceptable emulsions, microemulsions, solutions, suspensions,
syrups, and elixirs. In addition to an encapsulated or
unencapsulated conjugate, the liquid dosage forms may contain inert
diluents commonly used in the art such as, for example, water or
other solvents, solubilizing agents and emulsifiers such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
dimethylformamide, oils (in particular, cottonseed, groundnut,
corn, germ, olive, castor, and sesame oils), glycerol,
tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid
esters of sorbitan, and mixtures thereof. Besides inert diluents,
the oral compositions can also include adjuvants, wetting agents,
emulsifying and suspending agents, sweetening, flavoring, and
perfuming agents. As used herein, the term "adjuvant" refers to any
compound which is a nonspecific modulator of the immune response.
In certain embodiments, the adjuvant stimulates the immune
response. Any adjuvant may be used in accordance with the present
invention. A large number of adjuvant compounds is known in the art
(Allison Dev. Biol. Stand. 92:3-11, 1998; Unkeless et al. Annu.
Rev. Immunol. 6:251-281, 1998; and Phillips et al. Vaccine
10:151-158, 1992).
[0122] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the encapsulated or unencapsulated conjugate is mixed with at least
one inert, pharmaceutically acceptable excipient or carrier such as
sodium citrate or dicalcium phosphate and/or (a) fillers or
extenders such as starches, lactose, sucrose, glucose, mannitol,
and silicic acid, (b) binders such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,
sucrose, and acacia, (c) humectants such as glycerol, (d)
disintegrating agents such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate, (e) solution retarding agents such as paraffin, (f)
absorption accelerators such as quaternary ammonium compounds, (g)
wetting agents such as, for example, cetyl alcohol and glycerol
monostearate, (h) absorbents such as kaolin and bentonite clay, and
(i) lubricants such as talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof. In the case of capsules, tablets, and pills, the dosage
form may also comprise buffering agents.
[0123] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings and other
coatings well known in the pharmaceutical formulating art.
[0124] It will be appreciated that the exact dosage of the
inventive conjugate is chosen by the individual physician in view
of the patient to be treated. In general, dosage and administration
are adjusted to provide an effective amount of the inventive
conjugate to the patient being treated. As used herein, the
"effective amount" of an inventive conjugate refers to the amount
necessary to elicit the desired biological response. As will be
appreciated by those of ordinary skill in this art, the effective
amount of inventive conjugate may vary depending on such factors as
the desired biological endpoint, the drug to be delivered, the
target tissue, the route of administration, etc. For example, the
effective amount of inventive conjugate containing an anti-cancer
drug might be the amount that results in a reduction in tumor size
by a desired amount over a desired period of time. Additional
factors which may be taken into account include the severity of the
disease state; age, weight and gender of the patient being treated;
diet, time and frequency of administration; drug combinations;
reaction sensitivities; and tolerance/response to therapy.
[0125] The conjugates of the invention may be formulated in dosage
unit form for ease of administration and uniformity of dosage. The
expression "dosage unit form" as used herein refers to a physically
discrete unit of conjugate appropriate for the patient to be
treated. It will be understood, however, that the total daily usage
of the compositions of the present invention will be decided by the
attending physician within the scope of sound medical judgment. For
any conjugate, the therapeutically effective dose can be estimated
initially either in cell culture assays or in animal models,
usually mice, rabbits, dogs, or pigs. The animal model is also used
to achieve a desirable concentration range and route of
administration. Such information can then be used to determine
useful doses and routes for administration in humans. Therapeutic
efficacy and toxicity of conjugates can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., ED.sub.50 (the dose is therapeutically effective in 50% of
the population) and LD.sub.50 (the dose is lethal to 50% of the
population). The dose ratio of toxic to therapeutic effects is the
therapeutic index, and it can be expressed as the ratio,
LD.sub.50/ED.sub.50. Pharmaceutical compositions which exhibit
large therapeutic indices may be useful in some embodiments. The
data obtained from cell culture assays and animal studies can be
used in formulating a range of dosage for human use.
[0126] The present invention also provides any of the
above-mentioned compositions in kits, optionally with instructions
for administering any of the compositions described herein by any
suitable technique as previously described, for example, orally,
intravenously, pump or implantable delivery device, or via another
known route of drug delivery. "Instructions" can define a component
of promotion, and typically involve written instructions on or
associated with packaging of compositions of the invention.
Instructions also can include any oral or electronic instructions
provided in any manner. The "kit" typically defines a package
including any one or a combination of the compositions of the
invention and the instructions, but can also include the
composition of the invention and instructions of any form that are
provided in connection with the composition in a manner such that a
clinical professional will clearly recognize that the instructions
are to be associated with the specific composition.
[0127] The kits described herein may also contain one or more
containers, which may contain the inventive composition and other
ingredients as previously described. The kits also may contain
instructions for mixing, diluting, and/or administrating the
compositions of the invention in some cases. The kits also can
include other containers with one or more solvents, surfactants,
preservative and/or diluents (e.g., normal saline (0.9% NaCl), or
5% dextrose) as well as containers for mixing, diluting or
administering the components in a sample or to a subject in need of
such treatment.
[0128] The compositions of the kit may be provided as any suitable
form, for example, as liquid solutions or as dried powders. When
the composition provided is a dry powder, the composition may be
reconstituted by the addition of a suitable solvent, which may also
be provided. In embodiments where liquid forms of the composition
are used, the liquid form may be concentrated or ready to use. The
solvent will depend on the conjugate and the mode of use or
administration. Suitable solvents for drug compositions are well
known, for example as previously described, and are available in
the literature. The solvent will depend on the conjugate and the
mode of use or administration.
[0129] The invention also involves, in another aspect, promotion of
the administration of any of the conjugates described herein. In
some embodiments, one or more compositions of the invention are
promoted for the prevention or treatment of various diseases such
as those described herein via administration of any one of the
compositions of the present invention. As used herein, "promoted"
includes all methods of doing business including methods of
education, hospital and other clinical instruction, pharmaceutical
industry activity including pharmaceutical sales, and any
advertising or other promotional activity including written, oral
and electronic communication of any form, associated with
compositions of the invention.
[0130] The following examples are intended to illustrate certain
embodiments of the present invention, but do not exemplify the full
scope of the invention.
EXAMPLE 1
[0131] This example illustrates polymer synthesis of a triblock
polymer according to one embodiment of the invention. Carboxylic
end modified high molecular weight PLGA (with inherent viscosity of
0.6 dL/g in HFIP, hexafluoro-2-propanol) was purchased from
Absorbable Polymers International. Bifunctional PEG
(NH.sub.2--PEG-COOH) was purchased from Nektar Therapeutics. Amine
modified PSMA aptamer was purchased from RNA-Tec NV (Leuven,
Belgium). All other reagents were purchased from Sigma Aldrich.
[0132] The conjugation of PLGA and PEG were achieved in the
presence of EDC and NHS. Briefly, PLGA particles were dissolved in
acetonitrile. The carboxylic end of PLGA was activated by mixing
with NHS and EDC at a molar ratio of COOH to EDC and NHS and
stirred overnight at room temperature. The excess EDC and NHS in
the solution were quenched by adding 2-mercaptoethanol. The NHS
activated PLGA was purified by precipitation in a solution
containing ethyl ether and methanol, and followed by centrifugation
at 3000 g for 10 minutes. To conjugate the amine end of
NH.sub.2--PEG-COOH with the NHS-activated PLGA, both polymers mixed
at a molar ratio of 1:1.3 (PLGA-NHS:NH.sub.2--PEG-COOH) at room
temperature overnight. The resulting PLGA-PEG-COOH copolymer was
purified by precipitation in ethyl ether-methanol solution.
[0133] To make PLGA-PEG-aptamer triblock copolymer, PLGA-PEG
copolymers were first dissolved in acetonitrile, and aptamers were
dissolved in DMSO (dimethylsuloxide) and formamide. The conjugation
of the carboxylic end of COOH-PEG-PLGA and the amine end of aptamer
was done using the same EDC/NHS reaction as described for the
PLGA-PEG conjugation reaction.
[0134] The nanoparticles were formed by precipitating the triblock
copolymer in water. Briefly, the triblock polymer was dissolved in
acetonitrile at a concentration between 1 to 50 mg/mL, and then
added drop by drop in water. The nanoparticles formed instantly
upon mixing. The residual acetonitrile in the suspension was
evaporated by continuously stirring the suspension at room
temperature for 4 hours, followed by washing and rinsing the
nanoparticles using ultracentrifugation.
EXAMPLE 2
[0135] This example illustrates the synthesis of a multi-block
polymer for the development of small scale particles (i.e. pico,
nano or microparticles) that have a predetermined functionalized
surface for applications such as targeting certain cells, tissues,
or organs of the body; and may have the ability of minimizing host
immunogenicity by the presence of "stealth" materials on the
particle surface which may be a part of the multi-block copolymer;
and may have the ability of releasing pharmaceutical drugs at a
sustained rate. These small particles may have utility as research
tools or for clinical applications for targeting certain cells,
tissues, or organs for diagnostic, therapeutic, or a combination of
diagnostic and therapeutic applications. This example also shows
the pre-functionalization of the polymer by a biomacromolecule
block before nanoparticle formulation.
[0136] This example illustrates a technology platform for
synthesizing multi-block polymer that can be used for formulating
functionalized particle drug delivery system with unique abilities
such as targeting. The multi-block polymer includes a unique
targeting molecule that can bind to its specific receptor, and a
biodegradable polymeric material that can release bioactive drugs
at a sustained rate upon administration; and may also include a
third utility, i.e., a "stealth" material that can minimize the
host immunogenicity and/or increase the circulation half-life.
Additional molecules can be integrated into the block such as
fluorescent agents to develop targeted fluorescent particles for
combined imaging and therapeutic applications. The particles may be
generated at a nanoscale size by precipitation of the multiblock
copolymer in an aqueous medium. Taking advantage of the differences
in hydrophilicity and hydrophobicity of the distinct polymers
within the multi-block co-polymer system, nanoparticles can be
synthesized that can encapsulate a drug in the hydrophobic
component of the multi-block system forming the core of the
nanoparticle, while the hydrophilic targeting biomacromolecule
polymer, such as polynucleotides (i.e. aptamers) or polypeptides,
with or without a polyalkylene glycol polymer will be present on
the surface of the nanoparticle, resulting in a single step
synthesis or targeted nanoparticles.
[0137] Such nanoparticles can reach their targets after skin,
subcutaneous, mucosal, intramuscular, ocular, systemic, oral, or
pulmonary administration. The target tissue may represent
generalized systemic absorption or more specifically, targeting of
particles to distinct cells, tissues, or organs of the body. One
specific use of such targeted nanoparticles may be in the field of
cancer. It is possible to achieve specific and efficient tumor
specific targeting by systemic administration of multi-block
polymers containing targeting molecules that are directed against
epitopes or antigens present on tumor cells. The success of this
approach may depend on the prospect of high throughput synthesis of
cancer targeted nanoparticles. By developing libraries of particles
with distinct but overlapping biophysical and/or chemical
characteristics, as shown in this example, nanoparticles with high
cancer cell target specificity can be screened with desired
anti-cancer drug release kinetics. Thus, the multi-block polymer
synthesis platform discussed in this example enables the targeted
molecules to conjugate with stealth and drug release polymers, and
subsequently forms the desired nanoparticles to a one step reaction
under ambient condition.
[0138] This example describes a platform technology that enables
conjugation of targeted ligand onto a biodegradable polymer and
formation of functionalized nanoparticles in as few as a one-step
reaction. The composition of the nanoparticle and its surface
property can be accurately quantified. The potential implication of
this invention is broadly important to the field of nanotechnology
and cancer. Further, this example relates to multi-block copolymers
and at least one component of the multi-block may be a
biomacromolecule for targeted delivery, such as polynucleotide or
polypeptide. This example describes the synthesis of a multi-block
co-polymer system for rapid synthesis of particles which may be
targeted with polypeptides or polynucleotides (i.e. aptamers).
[0139] The synthesis of a multi-block polymer is initiated by
conjugation of functionalized biodegradable polyesters with
chemical groups such as, but not limited to, malimide or carboxylic
acid for easy conjugation to one end of thiol, amine or similarly
functionalized polyethers. The conjugation of polymer esters and
polyethers will be performed in organic solvents such as but not
limited to dichloromethane, acetonitrile, chloroform,
dimethylformamide, tetrahydrofuran, and acetone. The other free end
of the polyether would be functionalized with chemical groups for
conjugation to a library of targeting molecules such as but limited
to polymer of nucleic acids, fatty acids, peptides, carbohydrates,
peptidoglycans, or glycopeptides. The polynucleotides may contain
unique RNA, DNA, or modified RNA or DNA fragments which are
generated using natural nucleotides or nucleotides having a
substitution of a functional group including but not limited to
2'-F, 2'-OCH.sub.3, 2'-NH.sub.3; or polynucleotides generated using
L-enantiomeric nucleotides. The conjugation reaction between the
targeting molecules and the poly-ester-ether copolymer is achieved
by adding the targeted molecules solublized in an organic solvent
such as but not limited to formamide, dimethylformamide, pyridines,
dioxane, and dimethysulfoxide, to a solution of copolymer of
poly(ester-ether) in the solvents described above. Following each
of the two conjugation reactions, unconjugated macromers are washed
away by precipitating the polymer of interest in solvents such as
but not limited to ethyl ether, hexane, methanol and ethanol.
Biodegradable and biocompatible polymer poly(lactide-co-glycolide)
(PLGA) and polyethylene glycol (PEG) can be used as a model for the
block copolymer of poly(ester-ether). In one example, a
nuclease-stable oligonucleotide can be used for prostate specific
membrane antigen (PSMA) aptamer as the targeting molecule to
prostate cancer cells. Carboxylic acid modified PLGA (PLGA-COOH)
can be conjugated to the amine modified heterobifunctional PEG
(NH.sub.2--PEG-COOH) and form a copolymer of PLGA-PEG-COOH. By
using an amine modified PSMA aptamer (NH.sub.2-Aptamer), a triblock
polymer of PLGA-PEG-Aptamer can be obtained by conjugating the
carboxylic acid end of PEG and amine functional group on the
aptamer. The multiblock polymer can also be useful for imaging and
diagnostic applications. In such embodiment, a photo-sensitive or
environmental-responsible compound will be linked to the multiblock
polymer.
[0140] The targeted nanoparticles are formed by precipitation of
the multi-block polymer in an aqueous environment, in this example.
The nanoparticle formulation system described here is compatible
with high throughput biological assays in order to test the
nanoparticles generated from the multi-block polymer. It is
possible to perform high throughput assays to determine cellular
update of nanoparticles with different surface properties in vivo.
For an example, a combinatorial method could be used by changing
the composition of the multi-block polymer and its mixing ratio in
the nanoparticle formulation and thereby generating a library of
distinct formulations of pegylated nanoparticle-aptamer
bioconjugates: nanoparticle size (by varying the molecular weight
of PEG in the multiblock polymer), surface charge (by mixing the
multiblock polymer formulation with PEG polymer with a terminal
carboxylic acid group COOH-PEG-PLGA), surface hydrophilicity (by
mixing multiblock polymer with linear or branched PEG polymers of
various molecular weight, and/or density of aptamers on
nanoparticle surface (by controlling the mixing ratios of
multiblock polymer and methoxy modified PEG (mPEG-PLGA) or
carboxylic acid modified PEG (PLGA-PEG-COOH) which can be
conjugated to NH.sub.2-modified aptamers)). This example of the
multi-block copolymer allows for a rapid and reproducible synthesis
of a library of nanoparticles for further evaluation and
characterization.
EXAMPLE 3
[0141] This example illustrates determination of the amount of
aptamer expressed on the nanoparticle surfaces. To demonstrate that
a library of nanoparticles containing different ligand densities on
the nanoparticle surface can be formulated by diluting the
aptamer-PEG-PLGA triblock of Example 1 with diblock solution, the
triblock copolymer was serially diluted in PLGA-PEG diblock
copolymer then precipitated in water. To quantify the A10 aptamer
ligand density on the nanoparticle surface, the amide bond between
the A10 aptamer and the carboxyl functional group of PEG remained
on the nanoparticle surface was hydrolyzed in base, and the amount
of RNA aptamer recovered was quantified spectrophotometrically. It
was found that increasing the aptamer triblock in the nanoparticle
formulation linearly increased the amount of aptamer recovered from
the nanoparticle surface. For example, increasing the aptamer
triblock to diblock ratio from 0.02 to 0.10, the amount aptamer on
the nanoparticle surface increased from 100 micrograms to 450
micrograms. By comparing the amount of aptamer ligand recovered
from the nanoparticle surface with the total amount of aptamers
present in the NP formulation, it was found that increasing aptamer
triblock formulation increased the total proportion of aptamer that
can be expressed on the NP surface. By combining FIGS. 9A and 9B
with the in vitro and in vivo uptake study, the amount of ligand
surface density can be precisely controlled for the desired
therapeutic application. For example, based on the in vitro
nanoparticle uptake study and the in vivo study, it was found that
the 2% triblock formulation was sufficient to target LNCap prostate
cancer tumors with minimal amount of nonspecific uptake. Using
FIGS. 9A and 9B, it was determined that about 50% of the aptamer in
the 2% formulation was expressed on the nanoparticle surface, which
translates to approximately 100 micrograms of aptamer per mg of
aptamer.
[0142] The nanoparticles were prepared as follows. Nanoparticles
containing different amount of aptamer-PEG-PLGA triblock copolymer
and PLGA-PEG copolymer was precipitated in water. The aptamers on
the surface of the nanoparticles were hydrolyzed from the
nanoparticles were separated from the nanoparticles by
ultracentrifugation. Briefly, the nanoparticles were incubated in
10 mM KOH at 60.degree. C. for 30 minutes. The nanoparticle size
before and after base treatment was monitored by light scattering.
It was found that the nanoparticle diameter after the treatment
decreased by 6-8 nm. The aptamers removed from the nanoparticles
surface were separated from the nanoparticle core by
ultracentrifugation. The mass of the aptamers collected was
determined spectrophotometrically at UV absorbance at 260 nm. The
percentage of aptamer expressed on the nanoparticles surface was
determined by the amount of aptamer separated from the NP surface
divided by the total amount of aptamers triblock copolymer used in
the NP formulation.
EXAMPLE 4
[0143] In this example, a well-characterized nanoparticle
formulation is illustrated with physicochemical properties, drug
release kinetics and stability characteristics suitable for use in
the clinic. Here, a chemotherapeutic drug encapsulated
biodegradable nanoparticles is shown. The composition of the
targeted nanoparticles is made of the following four components:
poly(lactic-co-glycolic acid) (PLGA), an FDA approved controlled
release polymer system that can encapsulate a drug and release it
over time (properties that can be mediated by both drug diffusion
and polymer degradation); poly(ethylene glycol) (PEG), an FDA
approved polymer that can increase the circulating half-life of
nanoparticles; the drug docetaxel (Dtxl), an FDA approved
chemotherapeutic that is widely used in clinical practice; and a 56
base pair A10 2'-fluoropyrimidine nuclease stabilized RNA aptamer
(referred to as Aptamer) that can bind with high affinity and
specificity to Prostate Specific Membrane Antigen (PSMA), which is
significantly up-regulated on the surface of PCa cells and on many
tumor microvasculatures, and is recycled from cell surface at a
basal rate, allowing for the uptake of nanoparticle-aptamer
bioconjugates into the target cells.
[0144] In this example, the PLGA segment on the triblock copolymer
serves as the biodegradable matrix used to encapsulate drugs; the
PEG segment provides an attachment point while also enhances
circulating half-life; and the A10 aptamer segment represents the
PSMA specific targeting molecule. A PLGA-PEG-Apt triblock copolymer
was synthesized which can self assemble in water to form drug
encapsulated nanoparticles with functionalized molecules on the
surface of nanoparticles. Thus, an aptamer-PEG-PLGA triblock
copolymer system that enables conjugation of aptamers directly onto
the PLGA-PEG diblock copolymer before nanoparticle formation is
illustrated. The PLGA segment on the triblock copolymer serves as
the biodegradable drug delivery matrix that can be used to
encapsulate the drugs to be released at the targeted site; the PSMA
aptamer segment represents the PSMA specific targeting molecule;
and the PEG enhances circulating half-life by minimizing passive
non-specific uptake of particles. This technology of making an
aptamer-PEG-PLGA triblock copolymer can be used as a platform
technology is potentially suited to develop large scale production
of prefunctionalized nanoparticles while minimizing production time
and the nanoparticle batch to batch variation observed in the
post-particle surface modification. Another advantage of the making
targeted nanoparticles using triblock copolymer is to enable high
throughput synthesis of a large batch of prefunctionalized
nanoparticles with different surface and chemical properties by
simply mixing the triblock copolymer with other polymers containing
the desired properties.
[0145] Here, a library of nanoparticles with hydrodynamic radius
was generated by using different compositions of PLGA-PEG diblock
copolymer. As shown in the 3D plot in FIG. 10, the nanoparticle
size was directly proportional to the PLGA-PEG molecular weight,
whereby increasing the PLGA and/or PEG molecular weight increased
the size of nanoparticles. The nanoparticle size was more sensitive
to PLGA molecular weight than to PEG. The functional group on the
PLGA and PEG terminal end had no influence on the nanoparticle
size. Based on this plot, appropriate types of PLGA-PEG copolymer
can be chosen for mixing with aptamer triblock copolymer to
generate nanoparticles with different particle size.
[0146] Details of the synthesis process are as follows. Carboxylic
end modified high molecular weight PLGA (with inherent viscosity of
0.6 dL/g in HFIP) was purchased from Absorbable Polymers
International. Bifunctional PEG (NH.sub.2--PEG-COOH) was purchased
from Nektar Therapeutics. Amine modified PSMA aptamer was purchased
from RNA-Tec NV (Leuven, Belgium). All other reagents were
purchased from Sigma Aldrich.
[0147] The conjugation of PLGA and PEG were achieved in the
presence of EDC and NHS. Briefly, PLGA particles were dissolved in
acetonitrile. The carboxylic end of PLGA was activated by mixing
with NHS and EDC at a molar ratio of COOH to EDC and NHS and stir
overnight at room temperature. The excess EDC and NHS in the
solution were quenched by adding 2-mercaptoethanol. The NHS
activated PLGA was purified by precipitation in a solution
containing ethyl ether and methanol, and followed by centrifugation
at 3000 g for 10 minutes. To conjugate the amine end of
NH.sub.2--PEG-COOH with the NHS-activated PLGA, both polymers mixed
at a molar ratio of 1:1.3 (PLGA-NHS:NH.sub.2--PEG-COOH) at room
temperature overnight. The resulting PLGA-PEG-COOH copolymer was
purified by precipitation in ethyl ether-methanol solution. To make
PLGA-PEG-aptamer triblock copolymer, PLGA-PEG copolymers were first
dissolved in acetonitrile, and aptamers were dissolved in
dimethylsulfoxide and formamide. The conjugation of the carboxylic
end of COOH-PEG-PLGA and the amine end of aptamer was done using
the same EDC/NHS reaction as described for the PLGA-PEG conjugation
reaction.
[0148] Nanoparticles were formed by precipitating the copolymer in
water. Briefly, the triblock polymer was dissolved in acetonitrile
at a concentration between 1 to 50 mg/mL, and then added drop by
drop in water. The nanoparticles formed instantly upon mixing. The
residual acetonitrile in the suspension was evaporated by
continuously stirring the suspension at room temperature for 4 hrs,
followed by washing and rinsing the nanoparticles by
ultracentrifugation. To encapsulate chemotherapeutic drugs into the
nanoparticle core, triblock copolymer was mixed with hydrophobic
chemotherapeutic drugs such as docetaxel and paclitaxel, and then
precipitated in water followed by the same purification steps as
described above.
EXAMPLE 5
[0149] This example demonstrates that binding of PEGylated
aptamer-nanoparticle bioconjugates to LNCaP cells was significantly
enhanced when compared to control pegylated nanoparticles lacking
the A10 aptamer (FIG. 7). Targeted nanoparticles were formulated by
mixing the aptamer triblock copolymer with different amount of
PLGA-PEG diblock copolymer (FIG. 4A). Nanoparticles were
coencapsulated with .sup.14C-radiolabelled paclitaxel. The
percentage of nanoparticles endocytosed by the PCa cells was
quantified by the amount of radioactivity of 14C detected in the
cells. In the case of PC3 prostate epithelial cells, which do not
express the PSMA protein, no measurable difference in binding was
observed between the bioconjugate and the control group (FIG.
11).
[0150] In LNCaP cells, which do express PSMA protein, the data
demonstrates significant enhancement in the binding of targeted
nanoparticles vs. the non-targeted nanoparticles in LNCaP cells. A
notable observation was a remarkably low binding efficiency of
nanoparticles in non-targeted nanoparticles to both PC3 and LNCaP
cells, presumably attributed to the presence of PEG group.
Additionally, this example also showed that nanoparticles developed
from tri-block copolymer using random nucleic acid molecules failed
to have any targeting effect and behaved similar to the
non-targeted nanoparticles.
[0151] FIG. 7 shows the effect of aptamer concentration on the
nanoparticle surface on the rate of nanoparticle endocytosis by the
PCa cells in vitro. LNCaP cells (which express the PSMA protein;
left bars) and PC3 cells (which do not express PSMA; right bars)
were grown in 6 well tissue culture plates in the presence of
nanoparticles with different aptamer concentration on their
surface. Each formulation was obtained by mixing PLGA-PEG-aptamer
triblock with different amount of PLGA-PEG diblock copolymer prior
to particle formation. The PSMA cell specific uptake was quantified
after 4 hours using nanoparticles which had Dtxl and trace amount
of .sup.14C-paclitaxel encapsulated within them (note that
.sup.14C-Dtxl is not commercially available and vehicle
optimization was carried out with trace .sup.14C-paclitaxel
together with Dtxl) (n=5). The data was significant in that with as
little as 0.1% triblock in the formulation of nanoparticles we
begin seeing targeting effects, and this targeting effect was
largely plateaued after using 5% triblock in the formulation.
Additional use of triblock after this point results in the masking
of the PEG on the nanoparticle surface which can accelerate
clearance of particles and make the particles less "stealth."
[0152] In this example, to demonstrate the cellular uptake of
triblock nanoparticles was aptamer mediated, nanoparticles made
with various proportion of triblock copolymer were seeded onto
LNCap (PSMA+) and PC3 (PSMA-) cells. Briefly, 500,000 LNCap and PC3
cells were seeded to a 6-well tissue culture plate and incubated at
37.degree. C. overnight. Nanoparticles with different
concentrations of aptamers on the particle surface were formulated
by diluting aptamer triblock copolymers with PLGA-PEG diblock
copolymer. The nanoparticles were coencapsulated with docetaxel and
.sup.14C radiolabelled paclitaxel. The percentage of nanoparticles
endocytosed by the PCa cells was quantified by the amount of
radioactiviy of .sup.14C detected in the cells. The controls were
nanoparticles made without aptamer triblock copolymer, and
nanoparticles made using a nontargeting DNA triblock copolymer. The
nanoparticle formulations were seeded onto LNCaP cells and PC3
cells for two hours at 37.degree. C. The cells were then rinsed
3.times. with PBS to remove unbound nanoparticles from the culture
media. To quantify nanoparticle uptake, cells were trypsinized and
collected in a glass vial containing scintillation cocktail. The
amount of radioactivity in the cells collected were detected by
using a TriCarb scintillation counter.
EXAMPLE 6
[0153] This example shows in vivo targeting LNCap tumor cells in
mice. Human xenograft prostate cancer tumors were induced in 8-week
old balb/c nude mice (Charles River Laboratories, Wilmington,
Mass., USA). Mice were injected subcutaneously in the right flank
with 4 million LNCaP cells suspended in a 1:1 mixture of media and
matrigel (BD Biosciences, Franklin Lakes, N.J., USA). Prior to use
in tumor induction, LNCaP cells were cultured in RPMI-1640 medium
supplemented with 10% fetal bovine serum, 100 units/mL penicillin
G, and 100 mg/mL streptomycin. Tumor targeting studies were carried
out after the mice developed 100 mg tumors. 20 mice were randomly
divided into groups of four (5% aptamer triblock nanoparticles, 2%
aptamer triblock nanoparticles, 5% nonfunctional DNA triblock,
nanoparticles without triblocks). For intratumoral injections, mice
were anesthetized by intraperitoneal injection of avertin (200
mg/kg body weight), and dosed with different nanoparticle
formulations intratumorally. For systemic administrations,
nanoparticles were administered by retro-orbital injection. The
nanoparticles were traced by encapsulating .sup.14C-paclitaxel
prior to administration. Different groups were euthanized at 24 h,
and 200 mL of blood was drawn by cardiac puncture from each mouse.
The tumor, heart, lungs, liver, spleen and kidneys were harvested
from each animal. The .sup.14C content of tissues was assayed in a
Packard Tri-Carb Scintillation Analyser (Downers Grove, Ill., USA).
The tissues were solubilized in Solvable (Packard), and activity
was counted in Hionic-Fluor scintillation cocktail (PerkinElmer,
Boston, Mass., USA). The liver from each mouse was homogenized due
to its large size, and 100 mg of tissue was placed in a
scintillation vial for analysis. The other organs were placed
directly in scintillation vials. Each organ was solubilized in 2 mL
solvent for 12 h at 60.degree. C., and the resulting solution was
de-colored with 200 mL hydrogen peroxide for 1 h at 60.degree. C.
For the blood, 400 mL Solvable was added, and the vials were
otherwise treated similarly to the tissues. To determine 100% dose,
vials of the formulated nanoparticles were counted along with the
tissues. Data is presented as percent injected dose per gram of
tissue.
[0154] As can be seen in FIG. 8A, the nanoparticles were highly
targeted to the tumor in these mice with intratumoral injunction,
although less targeting was seen with systemic injection (FIG. 8B).
FIG. 8C shows the percentage of injected dose per gram of tissue as
a function of aptamer formulation.
[0155] Referring now to FIG. 11, to examine to effect of aptamer
surface density on targeting prostate cancer tumor in vivo, a
biodistribution study using radiolabelled nanoparticles was
conducted. The nanoparticle formulation was carried out according
to the FIG. 2B of the embodiment scheme slides. Briefly, aptamer
triblock was premixed with .sup.3H radiolabelled PLGA (component A
in FIG. 3B), and .sup.14C radiolabelled paclitaxel (lipophilic
drug). The radiolabelled PLGA and paclitaxel were used to trace the
biodistributions of the particles and the drugs, respectively.
Systemic administration of targeted nanoparticles with varying
concentration of triblock confirmed that maximal targeting of
nanoparticles was achieved with the 5% tri-block nanoparticle
formulation. Similar experiments performed with higher
concentration of the tri-block copolymer demonstrated a decrease in
tumor targeting as these particles are more susceptible to early
clearance presumably secondary to excessive masking of the PEG
layer on nanoparticle surface. Additionally, in other experiments
where nanoparticles were developed from tri-block copolymer using
random nucleic acid molecules, these failed to have any targeting
effect and behaved similar to non-targeted nanoparticles in vivo.
Another important finding was that increasing the aptamer ligand
expression on the nanoparticle surface increased nanoparticle liver
retention. This finding suggested that the aptamer ligand density
on the nanoparticle surface must be precisely controlled in order
to achieve tumor specific targeting while ensuring the nanoparticle
surface has enough stealth coating to bypass liver filtration.
[0156] While several embodiments of the present invention have been
described and illustrated herein, those of ordinary skill in the
art will readily envision a variety of other means and/or
structures for performing the functions and/or obtaining the
results and/or one or more of the advantages described herein, and
each of such variations and/or modifications is deemed to be within
the scope of the present invention. More generally, those skilled
in the art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the teachings of the present invention
is/are used. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. It is, therefore, to be understood that the foregoing
embodiments are presented by way of example only and that, within
the scope of the appended claims and equivalents thereto, the
invention may be practiced otherwise than as specifically described
and claimed. The present invention is directed to each individual
feature, system, article, material, kit, and/or method described
herein. In addition, any combination of two or more such features,
systems, articles, materials, kits, and/or methods, if such
features, systems, articles, materials, kits, and/or methods are
not mutually inconsistent, is included within the scope of the
present invention.
[0157] All definitions, as defined and used herein, should be
understood to control over dictionary definitions, definitions in
documents incorporated by reference, and/or ordinary meanings of
the defined terms.
[0158] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0159] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Multiple elements listed with "and/or" should be construed in the
same fashion, i.e., "one or more" of the elements so conjoined.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified. Thus, as a
non-limiting example, a reference to "A and/or B", when used in
conjunction with open-ended language such as "comprising" can
refer, in one embodiment, to A only (optionally including elements
other than B); in another embodiment, to B only (optionally
including elements other than A); in yet another embodiment, to
both A and B (optionally including other elements); etc.
[0160] As used herein in the specification and in the claims, "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, when separating items in a list, "or"
or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of," or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e. "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of,"
or "exactly one of." "Consisting essentially of," when used in the
claims, shall have its ordinary meaning as used in the field of
patent law.
[0161] As used herein in the specification and in the claims, the
phrase "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from any one or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
non-limiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0162] It should also be understood that, unless clearly indicated
to the contrary, in any methods claimed herein that include more
than one step or act, the order of the steps or acts of the method
is not necessarily limited to the order in which the steps or acts
of the method are recited.
[0163] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
* * * * *